Methods for producing insulin in response to non-glucose secretagogues

ABSTRACT

The present disclosure relates to the application of genetic engineering to provide artificial β cells, i.e. cells which can secrete insulin in response to glucose. This is achieved preferably through the introduction of one or more genes selected from the insulin gene, glucokinase gene, and glucose transporter gene, so as to provide an engineered cell having all three of these genes in a biologically functional and responsive configuration. Assays for detecting the presence of diabetes-associated antibodies in biological samples using these and other engineered cells expressing diabetes-associated epitopes are described. Also disclosed are methods for the large-scale production of insulin by perfusing artificial β cells, grown in liquid culture, with glucose-containing buffers.

The present application is a 371 of PCT/US92/04737, filed Jun. 2, 1992,and a continuation-in-part of U.S. Ser. No. 07/819,326, filed Jan. 13,1992 now U.S. Pat. No. 5,427,940; which is a continuation-in-part ofU.S. Ser. No. 07/710,038, filed Jun. 3, 1991 now abandoned; and furtherincludes subject matter that constitutes a continuation-in-part of U.S.Ser. No. 07/483,224, filed Feb. 20, 1990 now U.S. Pat. No. 5,175,085;the disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the preparation, culture anduse of engineered cells having the ability to secrete insulin inresponse to glucose, to methods for the detection of diabetes-associatedantigens, and to methods employing engineered cells in the production ofhuman insulin for use in, for example, type I diabetes mellitus. Inparticular aspects, the present invention relates to the growth ofengineered cells in liquid culture and the increase in glucose-mediatedinsulin release by such cells.

DESCRIPTION OF THE RELATED ART

Insulin-dependent diabetes mellitus (IDDM, also known as Juvenile-onset,or Type I diabetes) represents approximately 15% of all human diabetes.IDDM is distinct from non-insulin dependent diabetes (NIDDM) in thatonly IDDM involves specific destruction of the insulin producing β-cellsof the islets of Langerhans in the pancreas. The destruction of β-cellsin IDDM appears to be a result of specific autoimmune attack, in whichthe patient's own immune system recognizes and destroys the β-cells, butnot the surrounding α (glucagon producing) or δ (somatostatin producing)cells that comprise the islet.

The precise events involved in β-cell recognition and destruction inIDDM are currently unknown, but involve both the "cellular" and"humoral" components of the immune system. In IDDM, islet β-celldestruction is ultimately the result of cellular mechanisms, in which"killer T-cells" destroy β cells which are erroneously perceived asforeign or harmful. The humoral component of the immune system,comprised of the antibody-producing B cells, is also inappropriatelyactive in IDDM patients, who have serum antibodies against various βcell proteins. Antibodies directed against intracellular proteinsprobably arise as a consequence of β-cell damage which releases proteinspreviously "unseen" by the immune system. However, the appearance ofantibodies against several cell surface epitopes such as insulin,proinsulin, the "38kD protein", immunoglobulins, the 65kD heat shockprotein and the 64kD and 67kD forms of glutamic acid decarboxylase(GABA) are believed to be linked to the onset of IDDM (Lernmark, 1982).Antibodies in diabetic sera may also interact with the islet GLUT-2glucose transporter (Johnson, et al., 1990c).

A progressive loss of β-cell function is observed in the early stages ofNIDDM and IDDM, even prior to the autoimmune β cell destruction in IDDM.The specific function of glucose-stimulated insulin release is lost inislets of diabetic patients, despite the fact that such islets continueto respond to non-glucose secretagogues such as amino acids andisoproterenol (Srikanta, et al., 1983).

The participation of the pancreatic islets of Langerhans in fuelhomeostasis is mediated in large part by their ability to respond tochanges in circulating levels of key metabolic fuels by secretingpeptide hormones. Accordingly, insulin secretion from islet β-cells isstimulated by amino acids, three-carbon sugars such as glyceraldehyde,and most prominently, by glucose. The capacity of normal islet β-cellsto "sense" a rise in blood glucose concentration, and to respond toelevated levels of glucose (as occurs following ingestion of acarbohydrate containing meal) by secreting insulin is critical tocontrol of blood glucose levels. Increased insulin secretion in responseto a glucose load prevents chronic hyperglycemia in normal individualsby stimulating glucose uptake into peripheral tissues, particularlymuscle and adipose tissue.

Mature insulin consists of two polypeptide chains, A and B, joined in aspecific manner. However, the initial protein product of the insulingene in β-cells is not insulin, but preproinsulin. This precursordiffers from mature insulin in two ways. Firstly, it has a so-calledN-terminal "signal" or "pre" sequence which directs the polypeptide tothe rough endoplasmic reticulum, where it is proteolytically processed.The product, proinsulin, still contains an additional connecting peptidebetween the A and B chains, known as the C-peptide, which permitscorrect folding of the whole molecule. Proinsulin is then transported tothe Golgi apparatus, where enzymatic removal of the C-peptide begins.The processing is completed in the so-called secretory granules, whichbud off from the Golgi, travel to, and fuse with, the plasma membranethus releasing the mature hormone.

Glucose stimulates de novo insulin biosynthesis by increasingtranscription, mRNA stability, translation, and protein processing.Glucose also rapidly stimulates the release of pre-stored insulin. Whileglucose and non-glucose secretagogues may ultimately work through afinal common pathway involving alterations in K⁺ and CA⁺⁺ channelactivity and increases in intracellular CA⁺⁺ (Prentki, et al., 1987;Turk, et al., 1987), the biochemical events leading from changes in thelevels of a particular fuel to insulin secretion are initially diverse.In the case of glucose, transport into the β-cell and metabolism of thissugar are absolute requirements for secretion, leading to the hypothesisthat its specific stimulatory effect is mediated by, and proportionalto, its flux rate through glycolysis and related pathways (Ashcroft,1980; Hedeskov, 1980; Meglasson, et al., 1986; Prentki, et al., 1987;Turk, et al. 1987; Malaisse, et al., 1990). Strong support for this viewcomes from the finding that non-metabolizable analogues of glucose suchas 3-O-methyl or 2-deoxy glucose fail to stimulate insulin release(Ashcroft, 1980; Meglasson, et al., 1986).

A substantial body of evidence has accumulated implicating a specificfacilitated-diffusion type glucose transporter known as GLUT-2, and theglucose phosphorylating enzyme, glucokinase, in the control of glucosemetabolism in islet β-cells. Both proteins are members of gene families;GLUT-2 is unique among the five-member family of glucose transporterproteins in that it has a distinctly higher Km and Vmax for glucose.Glucokinase is the high Km and high Vmax counterpart of GLUT-2 among thefamily of hexokinases (Weinhouse, 1976). Importantly, both proteins haveaffinities for glucose that allow dramatic changes in their activitiesover the physiological range of glucose. This has led to the hypothesisthat these proteins work in concert as the "glucose-sensing apparatus"that modulates insulin secretion in response to changes in circulatingglucose concentrations by regulating glycolytic flux (Newgard, et al.,1990; Johnson, et al., 1990a).

In normal β-cells, glucose transport capacity is in excess relative toglycolytic flux. Thus, the GLUT-2 transporter likely plays a largelypermissive role in the control of glucose metabolism, while glucokinaserepresents the true rate-limiting step (Meglasson, et al., 1986;Newgard, et al., 1990). Implicit in this formulation, however, is theprediction that severe underexpression of GLUT-2 will result in loss ofglucose-stimulated insulin secretion in islets, an idea that hasrecently received strong experimental support from studies withspontaneous (Johnson, et al., 1990b; Orci, et al., 1990) as well asexperimentally induced (Chen, et al., 1990; Thorens, et al., 1990b)animal models of β-cell dysfunction, which have clear similarities tothe β-cell impairment observed in human NIDDM. Furthermore, RINm5Fclonal insulinoma cells derived from islet β-cells express GLUT-1, atransporter with a substantially lower Km and Vmax for glucose, as theirpredominant glucose transporter instead of GLUT-2. This may explain thefinding that the clonal cells fail to respond to glucose as an insulinsecretagogue (Thorens, et al., 1988).

Currently, there are significant deficiencies both in the diagnosis andtreatment of diabetes, particularly IDDM. For example, the most commonclinical diagnostic test, the oral glucose tolerance test (OGTT) suffersfrom severe drawbacks, such as subjective interpretation and the abilityto only identify individuals with advanced disease. The serological testfor cytoplasmic islet cell antibodies (ICA-cyt) (Bright, 1987;Gleichmann et al., i 1987) is a diagnostic procedure for detecting theonset of diabetes, which involves binding of patients' antibodies tocryostat sections of fresh human or primate pancreas. One evidentdisadvantage of this is the requirement for fresh human or primatetissue. Further difficulties are: false negatives (40%); subjectiveinterpretation; poor reproducibility; and the inability to detect cellsurface-directed antibodies which are known to specifically damage βcells (Doberson, et a1., 1980).

Even less progress has been made in developing new therapeuticstrategies for diabetics. Significant effort has been devoted to thestrategy of islet or pancreas fragment transplantation as a means forpermanent insulin replacement (Lacy, et al., 1986). However, thisapproach has been severely hampered by the difficulties associated withobtaining tissue, as well as the finding that transplanted islets arerecognized and destroyed by the same autoimmune mechanism responsiblefor destruction of the patients original islet β cells.

Treatment for diabetes is still centered around self-injection ofinsulin once or twice daily. Both recombinant and non-recombinantmethods are currently employed for the industrial production of humaninsulin for therapeutic use. Recombinant methods generally include theexpression of recombinant proinsulin in bacteria or yeast, followed bychemical treatment of the proinsulin to ensure correct disulfide bondlinkages between the A and B chains of the mature insulin molecule. Theproinsulin produced by microorganisms is processed to insulin by theaddition of proteolytic enzymes. Thereafter, the mature insulin peptidemust be purified away from the bacterial or yeast proteins, as well fromthe added proteases. The bacterial procedure involves 40 distinct steps.The non-recombinant methods typically include the purification of piginsulin from freshly isolated porcine pancreas or pancreatic islets.Each of the above methods suffer from the drawbacks of being technicallydifficult and laborious. The latter method is further complicated by thefact that the pancreas is a complex proteinaceous tissue with highlevels of active proteases that can degrade insulin and render itinactive as a hormone.

Accordingly, it is evident that improvements are needed both in thetreatment and diagnosis of diabetes and in the methods of insulinproduction for current therapeutic application.

SUMMARY OF THE INVENTION

The present invention is intended to address such disadvantages presentin the prior art. In general, the invention is based on the inventor'sdiscovery that recombinant DNA technology and cell culture methods maybe employed to engineer an "artificial β cell" that secretes insulin inresponse to glucose. The present invention provides a means of preparingartificial β cells that it is proposed can be employed in a variety ofapplications, such as, e.g., in the detection of diabetes-associatedantigens, in the clinical treatment of IDDM and even in the large-scaleproduction of correctly-folded insulin. In further aspects, the currentinvention provides methods for growing artificial β cells in liquidculture on gelatin beads and for the increased production of humaninsulin by perifusion of such recombinant cells with glucose-containingbuffers.

Turning first to embodiments directed to the recombinant engineering ofcells secreting insulin in response to glucose, it should be pointed outthat this aspect of the invention relates generally to an engineeredcell that includes a gene, preferably a recombinant gene, encoding afunctional glucose transporter protein, wherein the engineered cellssecrete insulin in response to glucose. This aspect of the invention isbased generally on the inventor's finding that where a cell is competentto secrete insulin generally, it may be converted to aglucose-responsive cell through the introduction of a gene encoding afunctional glucose transporter protein, such as a GLUT gene. For mostpurposes leading up to the ultimate treatment of the diabetic condition,one will desire to employ GLUT-2 as the recombinant glucose transportergene. This is because the GLUT-2 gene corresponds to that found andnormally expressed in β cells, and it is believed that this gene willultimately provide a more physiological response than other types ofglucose transporters.

As used herein, the term "engineered" or "recombinant" cell is intendedto refer to a cell into which a recombinant gene, such as a geneencoding a functional glucose transporter protein, has been introduced.Therefore, engineered cells are distinguishable from naturally occurringcells which do not contain a recombinantly introduced gene. Engineeredcells are thus cells having a gene or genes introduced through the handof man. Recombinantly introduced genes will either be in the form of acDNA gene (i.e., they will not contain introns), a copy of a genomicgene, genes produced by synthetic means, and/or genes positionedadjacent to a promoter not naturally associated with the particularintroduced gene.

Generally speaking, it will be more convenient to employ as therecombinant gene a cDNA version of the gene. It is believed that the useof a cDNA version will provide advantages in that the size of the genewill generally be much smaller and more readily employed to transfectthe targeted cell than will a genomic gene, which will typically be upto an order of magnitude larger than the cDNA gene. However, theinventor does not exclude the possibility of employing a genomic versionof a particular gene where desired.

Engineered cells of the present invention will generally be derived froma cell line comprised of cells capable of forming secretory granules.Secretory granules are generally confined to mammalian cells whose mainfunction is the synthesis and secretion of peptides. Generally speaking,secretory granules are found in endocrine cells. Secretory granules areformed by budding of intracellular membranous structures known as theGolgi apparatus. Polypeptide hormones are usually synthesized asprohormones and undergo proteolytic processing to yield the shorter,mature version of the hormone.

Thus, for example, the initial protein product of the insulin gene inβ-cells is preproinsulin. This precursor differs from mature insulin inthat it has a so-called "signal sequence" at its N-terminus, consistingof a stretch of hydrophobic amino acids that guide the polypeptide tothe rough endoplasmic reticulum. It also has a connecting peptidebetween the A and B chains that comprise the mature insulin molecule,this connector is known as the "C-peptide". The preproinsulin moleculeenters the lumen of the endoplasmic reticulum, in the process having itshydrophobic N-terminal "pre" region proteolytically removed. Theprocessed, correctly folded proinsulin molecule (still containing theC-peptide) is then transported to the Golgi apparatus. As the precursoris transported through the Golgi apparatus, enzymatic removal of theC-peptide connector begins.

Secretory granules are derived from Golgi membranes by a process ofbudding off and eventual separation. The resulting granule envelopes themixture of unprocessed proinsulin and the small amount of matureinsulin. Most of the processing of proinsulin to insulin occurs shortlyafter formation of the secretory granules by virtue of the fact that theenzymes responsible for this processing are found at highestconcentration within the granules. The granules are transported to theplasma membrane surface of the cell in response to secretory stimulisuch as glucose; whereupon they fuse with the plasma membrane andrelease their stores of the mature hormone. The important and uniquefeatures of this system are 1) the secretory granules allow a supply ofa particular hormone to be built up and stored for release at the timewhen it is needed to perform its function and 2) the presence ofprocessing enzymes in the granules allow efficient conversion of theprecursor forms of hormones to the mature forms. Cells that lacksecretory granules will thus likely not be useful for the purposes ofthis aspect of the invention.

Therefore, cells used in this aspect will preferably be derived from anendocrine cell, such as a pituitary or thyroid cell. Particularlypreferred endocrine cells will be AtT-20 cells, which are derived fromACTH secreting cells of the anterior pituitary gland, GH1 or the closelyrelated GH3 cells, which are derived from growth hormone producing cellsof the anterior pituitary, or other cell lines derived from this gland.AtT-20 cells are preferred for the following reasons. First, these cellshave been modified for insulin gene expression by stable transfectionwith a viral promoter/human proinsulin cDNA construct (this derivationof the AtT-20 cell line is known as AtT-20ins; both the parental AtT-20cell line and the insulin expressing AtT-20ins cell line are availablefrom American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md 20852). Second, AtT-20ins cells are able to process the proinsulinmRNA and preprotein to yield the correctly processed insulinpolypeptide. Third, their insulin secretory response to analogues ofcAMP compares favorably with the well-differentiated hamster insulinoma(HIT) cell line which is derived from hamster islet β-cells. Finally,studies from the inventor's laboratory have recently shown thatAtT-20ins cells contain significant amounts of the islet isoform ofglucokinase, making this the only tissue other than liver or islets inwhich glucokinase gene expression has been reported.

GH1 and GH3 cells were originally derived from the same batch of cellsisolated from a rat pituitary gland tumor. GH3 cells differ from GH1cells in that they secrete more growth hormone and also secreteprolactin (both lines are available from the American Type CultureCollection). These cells are believed to be preferred for the practiceof the invention because it has been shown that introduction of arecombinant preprosomatostatin gene into these cells results insecretion of the mature somatostatin peptide (Stoller, et al., 1989);Processing of the endogenous preprosomatostatin gene also occurs inδ-cells of the islets of Langerhans. The finding that an islet hormoneprecursor can be correctly processed in growth hormone secreting cellsof the anterior pituitary suggests that proinsulin processing will alsooccur in these cell, perhaps even more efficiently than in AtT-20inscells.

A number of cell lines derived from β-cells, commonly known asinsulinoma cells, are also preferred for the practice of this inventionand are readily available, particularly as concerns the therapeuticaspects of the work. For example, hamster insulinoma (HIT-T15) cells arewell studied and are readily available from the American Type TissueCollection. A number of rat insulinoma cell lines are also available.

The RINm5F and RINr1046-38 cell lines were derived from a radiationinduced tumor of the islet β-cells (Gazdar, et al., 1980; Clark, et al.,1990). MSL-G2 cells were derived from a liver metastasis of an isletcell tumor.

These cells require periodic passage in an animal host in order tomaintain expression of their endogenous insulin gene (Madsen, et al.,1988). Finally, the β-TC insulinoma cell line has been recently derivedfrom transgenic animals injected with a T-antigen gene driven by aninsulin promoter, resulting in specific expression of T-antigen in isletβ-cells and consequent immortalization of these cells (Efrat, et al.,1988).

RIN 1046-38 cells have been shown in the inventor's laboratory toexpress both GLUT-2 and glucokinase (Hughes, et al., 1991), and havebeen shown by Clark, et al. (1990) to be responsive to glucose. Glucosestimulation of insulin release from these cells is maximal at 0.5 mMglucose, however, a level far below the stimulatory concentration ofglucose required for insulin release from normal β-cells. Recent studiesin the inventor's laboratory have shown that this hypersensitivity toglucose in RIN 1046-38 cells may be due to high levels of hexokinaseactivity. Hexokinase performs the same function as glucokinase (glucosephosphorylation) but does so at much lower glucose concentrations(hexokinase has a Km for glucose of approximately 0.05 mM versus 8 mMfor glucokinase). It is proposed that lowering of hexokinase activity bymethods of recombinant DNA technology described below might make RINcells useful for the practice of this invention.

Of course, the type of engineering that will be required in order toachieve a cell that secretes insulin in response to glucose will dependon the property of the starting cell. In general, the inventor proposesthat in addition to the ability to form secretory granules, the abilityto functionally express certain genes is important. The functional genesthat are required include an insulin gene, a glucose transporter geneand a glucokinase gene. In the practice of the invention, one or more ofthese genes will be a recombinant gene. Thus, if the starting cell has afunctional insulin gene and a functional glucokinase gene, and thesegenes are expressed at levels similar to their expression in β-cells,but the cell does not have a functional glucose transporter gene,introduction of a recombinant glucose transporter gene will be required.Conversely, if the starting cell expresses none of the aforementionedgenes in a functional fashion, or at physiologic levels, it will benecessary to introduce all three. Since recombinant versions of allthree categories of genes are available to the art, and the specifictechnology for introducing such genes into cells is generally known, theconstruction of such cells will be well within skill of the art in lightof the specific disclosure herein.

As stated above, particularly preferred endocrine cells for use inaccordance with the present'invention are AtT-20_(ins) cells, which havebeen stably transfected to allow the production of correctly processedhuman insulin. Also as stated, it is generally preferred to employ theGLUT-2 isozyme to provide recombinant cells with a functional glucosetransporter. Engineered cells that combine both of these features havebeen created by the inventor, and one form of cell expressing highlevels of GLUT-2 mRNA, termed CTG-6 cells, are envisioned by theinventor to be of particular use in aspects of the present invention.

Where the introduction of a recombinant version of one or more of theforegoing genes is required, it will be important to introduce the genesuch that it is under the control of a promoter that effectively directsthe expression of the gene in the cell type chosen for engineering. Ingeneral, one will desire to employ a promoter that allows constitutive(constant) expression of the gene of interest. Commonly usedconstitutive promoters are generally viral in origin, and include thecytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat(LTR) sequence, and the SV40 early gene promoter. The use of theseconstitutive promoters will ensure a high, constant level of expressionof the introduced genes. The inventor has noticed that the level ofexpression from the introduced gene(s) of interest can vary in differentclones, probably as a function of the site of insertion of therecombinant gene in the chromosomal DNA. Thus, the level of expressionof a particular recombinant gene can be chosen by evaluating differentclones derived from each transfection experiment; once that line ischosen, the constitutive promoter ensures that the desired level ofexpression is permanently maintained. It may also be possible to usepromoters that are specific for cell type used for engineering, such asthe insulin promoter in insulinoma cell lines, or the prolactin orgrowth hormone promoters in anterior pituitary cell lines.

Certain particular embodiments of the invention are 30 directed toengineering cells with reduced hexokinase activity relative to the cellline from which it was prepared. There are four known isoforms ofhexokinase in mammals. Hexokinases I, II, and III have very low Kms(high affinities) for glucose, on the order of 0.05 mM. Hexokinase IV isglucokinase, which has a high Km for glucose of around 8-10 mM. In theislet β-cell, glucokinase is the predominant glucose phosphorylatingenzyme, while in most clonal cell lines grown in culture, the low Kmhexokinase I isoform predominates. The inventor proposes that expressionof hexokinases other than glucokinase at high levels in clonal cellsused for engineering will tend to make the cell glucose-responsive interms of insulin release at lower concentrations of glucose than isdesirable. Thus, it is proposed that the lower thehexokinase/glucokinase ratio, the more physiologic the insulin response.

Various approaches may be taken to reduce the hexokinase activity inengineered cells. one approach involves the introduction of an antisenseRNA molecule. Antisense RNA technology is now fairly well established,and involves the juxtaposition of the targeted gene in a reverseorientation behind a suitable promoter, such that an "antisense" RNAmolecule is produced. This "antisense" construct is then transfectedinto the engineered cell and, upon its expression, produces a RNAmolecule that will bind to, and prevent the processing/translation ofRNA produced by the targeted gene, in this case the hexokinase gene.

An alternative approach to the reduction of hexokinase action is througha technique known as positive/negative selection. This techniqueinvolves selection for homologous recombination of a hexokinase genesegment that renders the endogenous hexokinase gene nonfunctional.

In other embodiments, the present invention is directed to a method ofproviding a glucose-responsive insulin-secreting capability to a mammalin need of such capability. The method includes generally implantingengineered cells which secrete insulin in response to glucose into sucha mammal. It is proposed by the inventor that techniques presently inuse for the implantation of islets will be applicable to implantation ofcells engineered in accordance with the present invention. One methodinvolves the encapsulation of engineered cells in a biocompatiblecoating. In this approach, cells are entrapped in a capsular coatingthat protects the encapsulated cells from immunological responses, andalso serves to prevent uncontrolled proliferation of clonal engineeredcells. A preferred encapsulation technique involves encapsulation withalginate-polylysine-alginate. Capsules made employing this techniquegenerally contain several hundred cells and have a diameter ofapproximately 1 mm.

An alternative approach is to seed Amicon fibers with engineered cells.The cells become enmeshed in the fibers, which are semipermeable, andare thus protected in a manner similar to the micro encapsulates(Altman, et al., 1986).

After successful encapsulation or fiber seeding, the cells, generallyapproximately 1,000-10,000, may be implanted intraperitoneally, usuallyby injection into the peritoneal cavity through a large gauge needle (23gauge).

A variety of other encapsulation technologies have been developed thatare proposed by the present inventor will be applicable to the practiceof the present invention (see, e.g., Lacy et al., 1991; Sullivan et al.,1991; WO 9110470; WO 9110425; WO 9015637; WO 9002580; U.S. Pat. No.5,011,472; U.S. Pat. No. 4,892,538; WO 8901967, each of the foregoingbeing incorporated by reference). The company Cytotherapeutics hasdeveloped encapsulation technologies that are now commercially availablethat will likely be of use in the application of the present invention.A vascular device has also been developed by Biohybrid, of Shrewsbury,Mass., that may have application to the technology of the presentinvention.

In regard to implantation methods which may be employed to provide aglucose-responsive insulin-secreting capability to a mammal, it iscontemplated that particular advantages may be found in the methodsrecently described by Lacy et al. (Science, 254:1782-1784, 1991) andSullivan et al. (Science, 252:718-721, 1991), each incorporated hereinby reference. These concern, firstly, the subcutaneous xenograft ofencapsulated islets, and secondly, the long-term implantation of inlettissue in an "artificial pancreas" which may be connected to thevascular system as an arteriovenous shunt. These implantation methodsmay be advantageously adapted for use with the present invention byemploying engineered cells, as disclosed herein, in the place of the"islet tissue" of the prior art methods.

In still further embodiments, the present invention is directed tomethods of detecting the presence of diabetes-associated, or islet-celldirected, antibodies in a sample as a means of assessing the occurrenceor risk of diabetes onset. For uses in connection with diagnostic orantibody detection aspects of the present invention, it is contemplatedthat numerous additional types of engineered cells will prove to beimportant, particularly those which exhibit an epitope of a selectedantigen on their cell surface. Exemplary antigens include particularlyGLUT-2, and also glutamic acid decarboxylase (the 64KD islet antigen andthe less antigenic 67kD form), insulin, proinsulin, islet 38KD protein,65 kDa heat shock protein, selected immunoglobulins, insulin receptorsor other types of islet cell antigens, whether cytoplasmic or surface.However, it may be desirable to employ cells that do not secreteinsulin, in that antibody reactivity with insulin has been associatedwith false positive reactions.

Generally speaking, the cells are prepared by introducing genesexpressing relevant epitopes into cultured cell lines that can be grownin unlimited quantity. However, in the context of immunologically-baseddetection methods there is no requirement that the cells beglucose-response or have insulin-secreting capability. All that isrequired is that the these cells express on their surface an epitopeassociated either with the onset of diabetes or, more generally, anislet cell epitope. Furthermore, there is no requirement that the cellactually express the entire protein, in that all that is ultimatelyrequired is that the cell express an epitope that is recognized by theantibody that is sought to be detected. Therefore, the inventioncontemplates that subfragments which comprise antigenic epitopes may beemployed in place of the complete antigenic protein.

The first step of the detection methods of the invention will generallyinclude obtaining a biological sample suspected of containingdiabetes-associated or islet cell-directed antibodies. Generallyspeaking, the biological sample will comprise serum, plasma, blood, orimmunoglobulins isolated from such samples. However, the method will beapplicable to any sample containing antibodies, regardless of its sourceor derivation.

Next, the sample is contacted with an engineered cell expressing adiabetes-associated or islet cell-expressed epitope, under conditionseffective to allow the formation of an immunocomplex between theexpressed epitope and antibodies that may be present in the sample. Thisaspect is not believed to be particularly critical to the successfulpractice of the invention in that any incubation technique or conditionsthat favor immunocomplex formation may be employed. Preferred conditionsinclude incubation of the cells with serum in isotonic media such asphosphate buffered saline or Hanks balanced salt solution.

Lastly, the method is completed by testing for the formation of animmunocomplex between the diabetes-associated or islet cell epitopesexpressed by the cell and antibodies present in the sample, wherein apositive immunoreaction indicates the presence of the respectiveantibody in the sample. The testing method is not believed to be crucialto the overall success of the invention. Many types of testingprocedures for detecting immunocomplex formation are known in the artand are applicable, including RIA, EIA, ELISA, indirectimmunofluorescence, and the like. In general, all that is required is atesting/detection procedure that allows one to identify an interactionof immunoglobulins present in the sample and epitopes expressed on thesurface of the engineered cell.

Certain approaches to the foregoing method will provide particularadvantages. One such approach involves contacting the immunocomplexedcell with a molecule having binding affinity for the immunocomplexedantibody. The binding molecule is, generally speaking, any molecule thatis capable of binding the immunocomplexed antibody, and that isdetectable. Exemplary binding ligands include protein A,anti-immunoglobulin antibodies, protein G, or even complement.Preferably, the binding ligand includes an associated label that allowsfor the convenient detection of immunocomplexed antibodies. Typicallabels include radioactive materials, fluorescent labels, and enzymes.Often, one may achieve advantages through the use of an enzyme such asalkaline phosphatase, peroxidase, urease, β-galactosidase or others thatcan be detected through use of a colorimetric substrate.

Other specific embodiments may include the use of associating ligandssuch as biotin, which can complex with avidin or streptavidin andthereby bring the enzyme or other label into association with theantibody or binding ligand.

The detection of immunocomplexed cells through the use of a label may befurther improved, and even automated, through the application of cellsorter technology that can identify or quantify cells having associatedimmunocomplexed antibodies. Particularly preferred is the use of afluorescent label in conjunction with sorting of cells on afluorescence-activated cell sorter. The inventors have found that such asystem can screen 40-50 sera per hour using a singlefluorescence-activated cell sorter.

In other embodiments, one may simply employ a microscope slide testwherein cells are grown on polylysine coated slides, exposed to a testsample and then treated with an appropriate reagent capable of detectingimmunocomplex formation. The presence of complexes can then bedetermined by direct viewing in a microscope, especially when thedetecting reagent is an antibody that is labeled with a fluorescentmarker.

An extension of such embodiments concerns the delineation of thespecific epitope (or epitopes) within an antigenic protein, for exampleGLUT-2, that is recognized by antibodies in the sera of patients withdiabetes. It is proposed that mutant or chimeric protein molecules canbe constructed and expressed in recombinant AtT-20 cells, and used toinvestigate the binding of patients' antibodies, as described above. Thefailure of antibodies to bind to a mutant molecule after a specificdeletion, or likewise, the ability of antibodies to bind to a chimericmolecule after a specific insertion, would allow the identification ofthe diabetes-specific epitope. Candidate epitopes include multipleextracellular "loop" regions of the GLUT-2 molecule. Once such anepitope is identified, synthetic peptides corresponding to the specificregion of the protein sequence can be produced and used to developsimpler diagnostic procedures, for example, utilizing ELISAs or RIAs todetect the formation of an antibody/peptide complex.

It is further believed that the foregoing method may be employed as atechnique for selection of engineered clonal cells that express epitopesrecognized by autoantibodies. That is, one may prepare a series ofclones which comprise, for example, cDNA prepared to islet cell mRNA,express these DNAs in a recombinant cell and screen the resultantrecombinant cells with a known antibody composition to identify diabetesassociated antigens in addition to those specific antigens discussedabove.

In still further embodiments, the invention concerns a method fordetecting the presence of diabetes-associated antibodies in a biologicalsample, such as a sample of serum, plasma, blood, or in immunoglobulinsisolated therefrom. This method comprises contacting the samplesuspected of containing diabetes-associated antibodies with intactGLUT-2-expressing cells under conditions effective to allow theinteraction of any antibodies which may be present with GLUT-2, and thendetermining the degree of glucose uptake by the cells. Inhibition ofglucose uptake indicates the presence of diabetes-associated antibodiesin the sample.

Preferred cells for use in such embodiments are GLUT-2-expressingengineered cells, and particularly, GLUT-2-expressing AtT20_(ins) cells.Suitable conditions for assays of this kind include incubating the cellswith an IgG sample and determining the degree of glucose uptake using3-O-methyl-β-D-glucose.

Further important embodiments concern methods of using the engineeredcells of the present invention in the production of insulin, andparticularly, in the production of human insulin which can be used inthe treatment of IDDM. In certain aspects, the engineered artificial βcells are grown in culture and then contacted with a buffer containingglucose, thus stimulating the cells to produce and secrete insulin whichcan be collected and purified from the surrounding media. For use inconnection with this aspect of the present invention, CTG-6 engineeredcells are contemplated to be of particular use, but any cell prepared tosecrete insulin in response to glucose may be employed.

The inventor has discovered that a particularly useful approach to theproduction of human insulin in the above manner is theglucose-stimulation of artificial β cells grown in liquid culture. Assuch, the recombinant cells are contained within a column and subjectedto perfusion with a buffer at a physiological pH, such as Krebs Ringersalt (KRS) solution, pH 7.4. To stimulate the production and secretionof insulin, the column of cells is perifused with a glucose-containingbuffer, such as KRS, 5 mM glucose. At this stage, the insulin-containingeluent from the column is collected, which provides ideal startingmaterial for the purification of increased amounts of high-qualityinsulin for human use.

An alternative strategy for the isolation and purification of humaninsulin for use in IDDM therapy is to purify insulin directly from CGT-6cells or other GLUT-2 transfected AtT-20 cell lines. This is now aviable possibility as the present inventors have demonstrated thatGLUT-2 transfection causes an increase in intracellular insulin ofapproximately 5-fold in CGT-6 cells. These recombinant cells thuscontain sufficient insulin to enable the large scale production of humaninsulin from CGT-6-like calls possible.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Northern blot demonstrating the presence of GLUT-2 MRNA intissues and AtT-20ins cell lines. Each lane contains 6 μg of total RNA.Samples were prepared from liver, anterior pituitary and islet tissuesamples, as well as from untransfected (AtT-20ins) and GLUT-2transfected (AtT-20ins CGT-5 and CGT-6) AtT-20ins cell lines. The blotwas probed with radiolabeled antisense GLUT-2 cRNA, and as a control forgel loading, with an antisense oligonucleotide probe for 18S rRNA (Chen,et al., 1990).

FIG. 2. Immunoblot of GLUT-2 in tissues, untransfected cells (AtT-20ins)and cells transfected with the CMV/GLUT-2 construct (AtT-20ins CGT-5,-6).

FIG. 3A, FIG. 3B and FIG. 3C. Glucose transport into AtT-20ins cells.FIG. 3A, Measurements of 3-O-CH₃ glucose uptake as a function of glucoseconcentration for untransfected AtT-20ins cells (parental) and GLUT-2transfected lines CGT-5 and CGT-6. The symbol legend is shown in theupper left corner of this panel. FIG.3B: Reciprocal plot of glucoseuptake versus 3-O-CH₃ glucose concentration for GLUT-2 transfected linesCGT-5 and CGT-6. The calculated Km and Vmax values for glucose transportand the symbol legends are given in the upper left corner of the panel.FIG. 3C: Reciprocal plot of glucose uptake versus 3-O-CH₃ -glucoseconcentration for untransfected AtT-20ins cells (parental cell line).The calculated Km and Vmax values for glucose transport are indicated.Note the difference in the scales between panels B and C.

FIGS. 4A-4B. Insulin release for AtT-20ins cells in response to glucose,and glucose potentiation of forskolin induced secretion. FIG. 4A:Insulin release was measured from untransfected (AtT-20ins) and GLUT-2transfected (CGT-6) AtT-20ins lines incubated with varying glucoseconcentrations over the range of 0-2 OmM, or with 0.5 μM forskolin (F)or 0.5 μM forskolin +2.5 μM glucose (F+G) for a period of three hours.Data are normalized to the total cellular protein present in eachsecretion well and represent the mean ±SEM for 3-9 independentsecretions per well condition. *, p<0.001 compared to secretion at 0 mMglucose; #, p=0.002 compared to secretion at 0 mM glucose. FIG. 4B:Insulin release was measured from untransfected (AtT-20ins) and GLUT-2transfected (CGT-6) AtT-20ins lines incubated with 0.5 μM forskolin(Fors) and 2.5 mM glucose (G1c) in combinations indicated by the legend.Data are normalized to total cellular DNA in each secretion well and areexpressed as the mean ± SEM for 3-9 independent measurements.Statistically significant increases in secretion relative to the -G1c,-Fors control are indicated by the symbol * (p<0.001).

FIG. 5A and FIG. 5B. The utility of the FACS method for detecting thepresence of a specific immune complex. FIG. 5A: Graphs 1 and 2 arederived by treatment of GLUT-2 expressing AtT-20ins cells with theanti-GLUT-2 antibody X617 and treatment with anti-rabbit IgG secondantibody labeled with phycoerythrin. Graphs 3 and 4 represent cellsincubated with antibody X617 after it had been preincubated with GLUT-2expressing AtT-20ins cells. In FIG. 5B, a similar experiment wasperformed with parental AtT-20ins cells not expressing GLUT-2. In thesecells, no difference is seen between the naked antibody and antibodypreabsorbed with GLUT-2 expressing cells.

FIG. 6A, FIG. 6B and FIG. 6C. Preliminary data on patient serum. FIG. 6Ashows the fluorescence spectrum of GLUT-2 transfected AtT-20ins cellsincubated with the second antibody (phycoerythrin labeled anti-humanglobulin) alone. In FIG. 6B, the GLUT-2 transfected cells have beenincubated with serum isolated from a normal patient, resulting in ashift in the fluorescence intensity relative to the control in FIG. 6A.In FIG. 6C, cells are incubated with serum from a patient with new-onsetType I diabetes, resulting in an even greater shift.

FIG. 7A, FIG. 7B and FIG. 7C. Effects of IgG samples on glucose uptake.The effects of purified IgG from nondiabetic subjects (closed circles)and patients with new-onset IDDM (open circles) on the uptake of3-O-Methyl-β-D-Glucose by dispersed rat islet cells (FIG. 7A),GLUT-2-expressing AtT20_(ins) cells (FIG. 7B), and GLUT-1-expressingAtT20_(ins) cells (FIG. 7C) were determined. Data points for islet cellsand GLUT-2-expressing AtT20_(ins). cells are the mean (±SE) uptake of3-O-methyl-β-D-glucose by each cell type after incubation with purifiedIgG from 6 nondiabetic human sera and 7 new-onset IDDM patients. Datafor GLUT-1-expressing AtT20_(ins) cells were from 5 nondiabeticindividuals and 6 new-onset IDDM patients. The rate difference Betweencurves with IgG from nondiabetic sera and sera from IDDM patients inislet calls and GLUT-2-expressing AtT20_(ins) cells are significant atp<0.05.

FIG. 8. Specific IgG binding to GLUT-2 expressing AtT20_(ins) cells.Specific binding was determined by subtracting the percentage of cellsfound in R₂ using nontransfected atT20_(ins) cells from cells found inR₂ using GLUT-2-expressing AtT20_(ins) cells. Separation of thepopulation of IDDM patients from the nondiabetic population issignificant at p<0.0001.

FIG. 9. Insulin release from GLUT-1 versus GLUT-2 transfected AtT-20inscells during perifusion with glucose and forskolin. Approximately 50×10⁶cells were perifused at a flow rate of 0.5 ml/minute with HBBS buffercontaining 0 mM glucose (phases I, III, and V), 5 mM glucose (phases IIand IV), or 5 mM glucose +0.5 μM forskolin (phase VI). Effluent mediasamples were collected in 1.25 ml aliquots (every 2.5 minutes ) andassayed for insulin by radioimmunoassay. CGT-6, GLUT-2 transfectedAtT-20ins cells; GT1-15, GLUT-1 transfected AtT-20ins cells; AtT-20ins,parental cell line. The results of one typical experiment are shown.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

ENGINEERING OF "ARTIFICIAL" β CELLS

Insulin dependent diabetes mellitus (IDDM) is caused by autoimmunedestruction of insulin producing β-cells. Islet transplantation has beenextensively investigated as a strategy for curing IDDM, but suffers fromthe difficulties associated with procuring enough tissue. The presentinvention is based in part on the inventor's recognition that theproblem of islet supply could potentially be circumvented if a non-isletcell type could be engineered to secrete insulin in response tometabolic signals, since such cells could be grown in unlimited quantityin vitro. Such cells could ultimately replace daily insulin injectionsas therapy for Type I diabetes.

The participation of the pancreatic islets of Langerhans in fuelhomeostasis is mediated in large part by their ability to respond tochanges in circulating levels of key metabolic fuels by secretingpeptide hormones. Accordingly, insulin secretion from islet β-cells isstimulated by amino acids, three-carbon sugars such as glyceraldehyde,and most prominently, by glucose. While these diverse secretagogues mayultimately work through a final common pathway involving alterations inK+ and Ca++ channel activity and increases in intracellular Ca++(Prentki, et al., 1987; Turk, et al., 1987), the biochemical eventsleading from changes in the levels of a particular fuel to insulinsecretion are initially diverse. In the case of glucose, transport inthe β-cell and metabolism of this sugar are absolute requirements forsecretion, leading to the hypothesis that its specific stimulatoryeffect is mediated by and proportional to its flux rate throughglycolysis and related pathways (Ashcroft, 1980; Hedeskov, 1980;Meglasson, et al., 1986; Prentki, et al., 1987; Turk, et al., 1987;Malaisse, et al., 1990). Strong support for this view comes form thefinding that non-metabolizable analogues of glucose such as 3-O-methylor 2-deoxy glucose fail to stimulate insulin release (Ashcroft, 1980;Meglasson, et al., 1986).

A substantial body of evidence has accumulated implicating a specificfacilitated-diffusion type glucose transporter known as GLUT-2, and theglucose phosphorylating enzyme, glucokinase, in the control of glucosemetabolism in islet 62-cells. Both proteins are members of genefamilies; GLUT-2 is unique among the five-member family of glucosetransporter proteins (GLUTs 1-5; Bell, et al., 1990; Thorens, et al.,1990a) in that is has a distinctly higher Km and Vmax for glucosetransport. Glucokinase (also known as Hexokinase IV) is the high Km,high Vmax counterpart of GLUT-2 among the family of hexokinases(Weinhouse, 1976). Importantly, both proteins have affinities forglucose that allow dramatic changes in their activities over thephysiological range of glucose. This has led to the hypothesis thatthese proteins work in concert as the "glucose-sensing apparatus" thatmodulates insulin secretion in response to changes in circulatingglucose concentrations by regulating glycolytic flux (Newgard, et al.,1990; Johnson, et al., 1990a).

In normal β-cells, glucose transport capacity is in excess relative toglycolytic flux. Thus, the GLUT-2 transporter likely plays a largelypermissive role in the control of glucose metabolism, while glucokinaserepresents the true rate-limiting step (Meglasson and Matchinsky, 1986;Newgard, et al., 1990). Implicit in this formulation, however, is theprediction that severe underexpression of GLUT-2 will result in loss ofglucose-stimulated insulin secretion in islets, an idea that hasrecently received strong experimental support from studies withspontaneous (Johnson, et al., 1990b; Orci, et al., 1990) as well asexperimentally induced (Chen, et al., 1990; Thorens, et al., 1990b)animal models of β-cell dysfunction.

IDDM has traditionally been treated by insulin replacement, eitherclassically, by external administration, or experimentally, bytransplantation of islets or pancreas fragments. The latter strategy isnot likely to be broadly applicable because of the difficulty andexpense associated with the isolation of large numbers of islets. Thepresent invention is directed to an alternative approach, that of usingmolecular techniques to engineer an "artificial β-cell", i.e., anon-islet cell capable of performing glucose-stimulated insulinsecretion, which can be grown in unlimited quantity in vitro.

The anterior pituitary cell line AtT-20ins is preferred because ofimportant similarities to β-cells. First, these cells have been modifiedfor insulin gene expression by stable transfection with a viralpromoter/proinsulin cDNA construct (Moore, et al., 1983). Second,AtT-20ins cells are able to process the proinsulin mRNA and preproteinto yield the correctly processed insulin polypeptide. Third, theirsecretory response to analogues of cAMP compares favorably with the welldifferentiated hamster insulinoma (HIT) cell line (Moore, et al., 1983).Finally, AtT-20ins cells contain significant amounts of the isletisoform of glucokinase (Hughes, et al., 1991), making this the onlytissue other than liver or islets in which glucokinase gene expressionhas been reported.

On the other hand, AtT-20ins cells differ from islets in two importantways. First, they do not secrete insulin in response to glucose, andsecond, they express the low Km GLUT-1 glucose transporter mMNA and notGLUT-2 (Hughes, et al., 1991). The inventor hypothesized that the lackof glucose responsiveness in AtT-20ins cells could be explained eitherby deficient capacity or altered affinity of glucose uptake relative tonormal islets. To test this hypothesis, AtT-20ins cells were stablytransfected with GLUT-2 cDNA. Surprisingly, the inventor found thatcells engineered in this way gained glucose-stimulated insulin secretionand glucose potentiation of non-glucose secretagogue stimulation, albeitwith a dose-response curve that is different from normal islets.

Engineering of the AtT-20ins cells generally involved construction of asuitable GLUT-2 expression vector, transfection of AtT-20ins cells withthe vector, and selection of stable transfectants. To accomplish this,rat islet GLUT-2 cDNA (Johnson et al., 1990a) was cloned into the vectorpCB-7, a derivative of vector pCMV4 (Anderson, et al., 1989),immediately downstream of its cytomegalovirus (CMV) promoter. pCB-7 wasconstructed by Drs. Michael Roth and Colleen Brewer of the BiochemistryDepartment, University of Texas Southwestern Medical Center at Dallasand provided as a gift to the inventors. It differs from pCMV-4 in thatit contains a hygromycin resistance gene; thus, cells transfected withthe pCB7/GLUT-2 construct can be selected for stable integration of thevector DNA into the cell's genome by treatment with hygromycin.AtT-20ins cells were transfected with this construct usingelectroporation, and stable transfectants were selected with hygromycin.

Expression of GLUT-2 mRNA was evaluated by blot hybridization analysisof AtT-20ins cells, either transfected or untransfected with acytomegalovirus (CMV) promoter/GLUT-2 hybrid gene, and in extracts ofrat liver, islets of Langerhans, and anterior pituitary tissues. Aradiolabeled GLUT-2 antisense RNA probe (Johnson, et al., 1990a; Chen,et al., 1990) was hybridized to a blot containing equal amounts of RNAfrom four GLUT-2 transfected AtT-20ins cell lines (CGT-1, CGT-2, CGT-5,CGT-6), untransfected AtT-20ins cells, and the three primary tissues(FIG. 1). Steady state levels of GLUT-2 mRNA were highest in CGT-5 andCGT-6; the former contained approximately half as much and the latter anequal amount of GLUT-2 MRNA as rat islets, and they contained 10 and 16times as much, respectively, as rat liver, measured by densitometricscanning and normalization to the signal obtained with an 18S mRNAprobe. The transfected lines contained a smaller GLUT-2 transcript thanliver or islets (2.2 versus 2.8 kb) because 635 base paris of the 3'untranslated region were removed in the course of cloning the GLUT-2cDNA into the pCB-7 vector. Lines CGT-1 and CGT-2 exhibited less activeexpression of GLUT-2. Untransfected AtT-20ins cells and primary anteriorpituitary cells did not contain detectable amounts of GLUT-2 mRNA,consistent with the inventor's previous work (Hughes, et al., 1991).

In order to evaluate the levels and molecular status of the expressedGLUT-2 protein in transfected AtT-20ins cells, crude membrane fractionswere resolved by SDS/PAGE, the separated proteins transferred tonitrocellulose, and GLUT-2 protein detected with an antibody raisedagainst its C-terminal hexadecapeptide sequence (Johnson, et al.,1990b). The antibody recognized two distinct bands in liver and islets,with apparent molecular weights of 70 and 52 kd in liver and slightlydifferent sizes of 72 and 56 kd in islets (FIG. 2, left). Consistentwith the RNA blot hybridization data, untransfected AtT-20ins cells werefound to lack GLUT-2 protein, while a single intense band ofapproximately 70 kd was observed in extracts from either of thetransfected lines. The specificity of the antibody was demonstrated bythe fact that all bands were blocked by preincubation of the antibodywith the antigenic peptide (FIG. 2, right).

In light of the observed differences in molecular species of GLUT-2observed in the cell types studied, the distribution and sorting of theexpressed GLUT-2 proteins was evaluated in transfected AtT-20ins cellsby immunocytochemical analysis and light microscopy, using the sameantibody employed for blot hybridization analysis. In the lines withhighest GLUT-2 expression, abundant GLUT-2 expression was detected atthe cell membrane. The signal was entirely blocked by preincubation ofthe antibody with the antigenic peptide and was not seen inuntransfected cells or in cells transfected with the vector lacking theGLUT-2 insert. Thus, the transfected AtT-20ins cells not only had thecapacity to produce GLUT-2 mRNA and protein but also sort the protein tothe cell membrane, as occurs in both islets and liver (Thorens, et al.,1988; Tal, et al., 1990; Orci, et al., 1989, Orci, et al., 1990).

It was further found that the engineered lines with high levels ofGLUT-2 expression (CGT-5, CGT-6) transported glucose very rapidly, withan estimated Km for glucose of 18 mM and a Vmax of 19 mmoles/min/litercell space. In contrast, the untransfected parental AtT-20ins linetransported glucose much less efficiently, with an apparent Km forglucose of 2 mM and a Vmax of 0.5, consistent with its expression of theGLUT-1 mRNA (Hughes, et al., 1991), which encodes the low Km glucosetransporter found in most clonal cell lines (Flier, et al., 1987;Birnbaum, et al., 1987). The transfected AtT-20ins cells have glucosetransport kinetics that are remarkably similar to isolated, dispersedislets of Langerhans, which have a Km of 18 mM for glucose and a Vmax of24 mmoles/min/liter cell space (Johnson, et al., 1990a). Thus, theGLUT-2 cDNA clearly encodes the protein responsible for the high Kmglucose transport activity in islets and liver, and is capable oftransferring this activity in to the AtT-20ins cell line. The fact thatonly the larger protein species of GLUT-2 is detected in transfectedAtT-20ins cells indicates that it is an active glucose transporter.

Insulin secretion from GLUT-2 transfected and untransfected cells wasmeasured over a range of glucose concentrations from 0-20 mM. Inmeasuring glucose-stimulated insulin release from AtT-20ins cells andCGT-6 cells, glucose was found to have no significant effect on insulinrelease from parental AtT-20ins cells, consistent with previous results(Hughes, et al., 1991). AtT-20ins cells transfected with the pCB7 vectorlacking a GLUT-2 insert were also found to be unresponsive to glucose.GLUT-2 transfected cells, in contrast, were found to be clearly glucoseresponsive. A submaximal but statistically significant (p=0.002)increase in insulin release relative to insulin release at 0 mM glucosewas observed at the lowest concentration of glucose studied (5 μM);maximal stimulation of approximately 2.5-fold was observed at all higherconcentrations over the range 10 μM-20 mM (p<0.001). It is highlyunlikely that these results can be attributed to clonal selection ofglucose responsive subpopulations of the parental AtT-20ins cells, sincecells transfected with vector lacking GLUT-2 failed to respond, whiletwo independent GLUT-2 expressing lines (CGT-5 and CGT-6) gained glucosesensing.

In normal islets, glucose potentiates the insulin secretory response tovarious β-cell secretagogues, including agents that increaseintracellular cAMP levels (Ullrich and Wollheim, 1984; Malaisse, et al.,1984). The inventor therefore studied the potentiating effect of glucoseon insulin secretion in the presence of forskolin, dibutyryl cAMP, andIBMX. Glucose has a modest stimulatory effect on forskolin stimulatedinsulin release from parental AtT-20ins cells, when the data wereexpressed either as insulin release/mg cellular protein, or as insulinrelease/mg cellular DNA. In contrast, glucose had a powerfulpotentiating effect on forskolin stimulated insulin release fromtransfected CGT-6 cells. The response was unchanged by glucoseconcentration over the range of 1-5 mM, and similar potentiating effectsof glucose on dibutryl cAMP and IBMX induced secretion were alsoobserved.

Insulin secretion experiments involved static incubation of cells withthe secretagogue of interest for three hours, and thus provide littleinformation about the dynamics of insulin release. The inventor thusgrew the parental and transfected AtT-20ins cell lines on gelatin beadsin liquid culture, allowing their secretory properties to be studies byperfusion with glucose containing media. In this configuration, insulinwas released within minutes of the start of glucose perfusion, and thesecretion response exhibited a first and second phase as ischaracteristic of normal β-cells. Maximal stimulation of insulin releaseoccurred during the first 10 minutes of perfusion with glucose (firstphase) and was 10-fold greater than baseline (in the absence ofglucose).

A remarkable finding of this work is that transfection of AtT-20inscells with the GLUT-2 cDNA results in a substantial increase inintracellular insulin content, despite the fact that insulin-geneexpression is driven by the glucose insensitive Rous sarcoma viruslong-terminal repeat enhancer/promoter in these cells. Native AtT-20inscells and the GLUT-2 transfected CGT-6 cells were grown for 3 days inmedia supplemented with low (1 mM) or high (25 mM) glucose. The CGT-6cells were found to contain 3.6-fold and 5.4-fold more insulin than theAtT-20ins cells when studied at low and high glucose, respectively(p<0.001 for both comparisons). Furthermore, insulin content wasapproximately double in the CGT-6 cells grown at high glucose comparedwith the same cells grown at low glucose (p<0.001). In contrast, in theuntransfected AtT-20ins cells, high glucose caused only a 20% increasein insulin content.

Although the inventor has succeeded in engineering an AtT-20ins cellline with glucose-stimulated insulin secretion, maximal insulinsecretion from these cells occurs at a much lower glucose concentrationthan observed for normal islets, which do not respond at levels lessthan the fasting glucose concentration of approximately 4-5 mM, andwhich have not reached maximum secretion at the upper range ofphysiological glucose (10 mM). The potentiating effect of glucose onforskolin, dibutryl cAMP, or IBMX induced insulin secretion fromAtT-20ins cells is also maximal at low glucose. The heightenedsensitivity of GLUT-2 transfected AtT-20ins cells to both the direct andpotentiating effects of glucose is reminiscent of a number of cell linesderived from insulinoma (β-cell) tumors (Praz, et al., 1983; Halban, etal., 1983; Giroix, et al., 1985; Meglasson, et al., 1987; Clark, et al.,1990). For example, the rat insulinoma cell line RIN 1046-38 isresponsive to glucose when studied after short periods of time in cellculture (between passages 6-17), albeit with a maximal response atsub-physiological glucose levels, as in transfected AtT-20ins cells.With longer time in culture (passage number greater than 50), allglucose-stimulated insulin secretion is lost (Clark, et al., 1990). Lowpassage RIN 1046-38 cells contain both glucokinase and GLUT-2, but loseexpression of these genes when studied at higher passages.

The fact that both transfected AtT-20ins cells and RIN1046-38 cells oflow passage number respond to subphysiological levels of glucose,despite expression of glucokinase and GLUT-2, suggests that these cellsshare metabolic determinants that can override the regulatory functionof the high Km components. Given that the glucose transport kinetics ofnormal islets are recapitulated in GLUT-2 transfected AtT-20ins cells,the increased sensitivity of the clonal cells to glucose mightalternatively be explained by alteration in regulation of glucosephosphorylation. While hexokinase activity is readily measured in isletcell extracts, this enzyme is thought to be potently inhibited (by asmuch as 95%) inside the intact islet cell (Trus, et al., 1981; Giroix,et al., 1984). Thus, in the presence of stimulatory concentration ofglucose, normal islets have both sufficient glucokinase activity andinhibited hexokinase (the levels of glucose-6-phosphate, an inhibitor ofhexokinase, increase during glucose stimulation) to allow the control ofglucose metabolism to be tied directly to glucokinase activity (Km of˜10 mM in islets) (Meglasson, et al., 1986).

AtT-20ins cells have glucokinase activity, but it represents only 9% oftotal glucose phosphorylation in these cells, and only 32% of theactivity measured in normal islets (Table 1 in Example I below); theproportions of glucose phosphorylating enzymes in RIN1046-38 cells aresimilar to those found in AtT-20ins cells (Newgard, C. B., unpublishedobservations). Hexokinase I, the isoform that is expressed in mostclonal cell lines (Arora, et al., 1990) is found bound to mitochondriaand in a free cytosolic form (Lynch, et al., 1991); in the former state,the enzyme is less sensitive to glucose-6-phosphate inhibition (Wilson,1984). Thus, in addition to the fact that AtT-20ins cells have reducedglucokinase activity, they may also have altered regulation ofhexokinase such that it becomes the predominant glucose phosphorylatingenzyme at any concentration of glucose studied.

The increased sensitivity of GLUT-2 expressing AtT-20ins or RIN cellscan be explained as follows. Expression of the GLUT-2 transporter notonly increases the Km for transport, but also the transport capacity atall glucose concentrations studied. Our data show that at 2.5 mMglucose, for example, there is an approximately 10-fold increase inglucose uptake in the GLUT-2 transfected cells compared to the parentalline (see FIG. 3A). This means that even at glucose concentrations thatwould be sub-stimulatory for islets, transport into GLUT-2 transfectedAtT-20ins cells will be rapid and hexokinase activity (Km for glucose of˜0.01 mM) will be maximal, and the generation of glucose-relatedsecretory signals will be maximized at low glucose as a consequence. Theinventor is currently investigating whether the hexokinase:glucokinaseratio can be altered by molecular techniques in GLUT-2 transfectedAtT-20ins cells, and if so, whether a glucose dose-response curveresembling that of islets will be gained.

As discussed above, an imbalance in the hexokinase/glucokinase ratio mayat times result in maximal insulin secretory response atsubphysiological glucose concentrations. The inventor proposes that amore physiologic glucose response may be achieved by "knocking out"hexokinase activity in engineered cells of the present invention. Oneapproach is to co-transfect these cells with antisense hexokinaseconstructs. This can be achieved, for example, using the CMV vectorsystem described for GLUT-2 transfection, with the exception that theplasmid will contain an alternate resistance gene, such as puromycin orhistidinol, since the AtT-20ins cell line is resistant to both neomycin(due to stable integration of the SV40-insulin-neo chimeric construct)and hygromycin (due to stable integration of the CMV-GLUT-2-hygromycinchimeric construct). Recently, the hexokinase isozyme expressed in mousehepatoma cells has been cloned and characterized (Arora, et al., 1990)and shown to be approximately 92% identical to the hexokinase Isequences derived from rat brain (Schwab, et al., 1989) and human kidney(Nishi, et al., 1988).

In order to generate antisense probes with exact sequence identity tothe homologue of hexokinase I being expressed to the engineered cell,the hexokinase variant present in the cell was converted to cDNA byreverse transcribing the MRNA and amplification of the DNA product, aprocedure recently employed in the inventor's laboratory foramplification of glucokinase MRNA from islets, RIN cells, AtT-20inscells, and primary anterior pituitary cells (Hughes, et al., 1991). Theoligonucleotides used for amplification were based on the publishedsequence of the mouse hepatoma hexokinase I (Arora, et al., 1990). Theoligonucleotides included restriction enzyme recognition sequences attheir 5' ends to facilitate directional cloning of the amplified cDNAinto the selected vector in an antisense orientation.

Because the vector contains both the transcription termination andpolyadenylation signal sequences downstream of the cloning cassette,processing of the antisense transcripts should proceed normally. Proofof this comes from the data derived with the GLUT-2/CMV construct, whichwas prepared using a restriction site in the 3' untranslated region ofthe GLUT-2 cDNA that removed some 600 bases of the 3' tail, a maneuverthat had no effect on transcription or translation of the GLUT-2 mRNA.One may desire to select various, different portions of the hexokinasesequence, since various investigators have reported success withantisense technology with full length antisense messages, or partialtranscripts that either target the ATG initiator codon and surroundingsequence or the 3' untranslated region and poly A tail (Walder, 1988).

It is proposed that engineered lines may be transfected with antisenseconstructs by electroporation. After appropriate selection to obtaincolonies that have stably integrated the antisense hexokinase constructinto their genome, expression of the antisense mRNA can be evaluated byhybridization to labeled sense RNA, e.g., prepared with the pGEM vectorsystem (Promega). Blot hybridization analysis may be carried out notonly with the probe corresponding to the antisense construct, but toregions outside as well, since cellular factors known to unwind RNA: RNAduplexes results in modification of that RNA, thus interfering with itsdetection on Northern blots (Walder, 1988). One may assess whether thepresence of antisense mRNA is capable of affecting the level ofhexokinase protein(s) through the use of antibodies against relevanthexokinase sequences).

Should the foregoing general antisense approach fail to provide adequatehexokinase suppression in the particular system selected, modifiedantisense oligonucleotides may be employed. For example, an antisenseoligonucleotide may be prepared to sequences surrounding and/orcontaining the ATG initiation codon, for example, and introduced intocells by simply incubating the cells in media containing theoligonucleotide at high concentration. This approach bypassesuncertainties about the stability of longer antisense hexokinasetranscripts synthesized from the construct and should providesuppression of hexokinase activity for a period of time sufficient toassess the functional consequences. On the negative side, theoligonucleotide antisense procedure can only cause a transient reductionin endogenous expression, and is thus not applicable to the engineeringof a stable "artificial" β-cell.

A second alternative that bypasses the issue of effectiveness ofantisense strategies altogether would be to knock out the endogenoushexokinase gene of interest cells using a positive/negative selectionprotocol (Mansour, et al., 1988; Capecchi, 1989; Zheng, et al., 1990) toselect for homologous recombination of a hexokinase gene segment thatrenders the endogenous hexokinase gene nonfunctional. This approachinvolves cloning of at least a segment of the hexokinase gene(s)expressed in the engineered cells either by library screening or PCR™amplification, and construction of a vector that contains a genomicfragment, preferably containing exons that encode the putative ATP orglucose binding sites (Arora, et al., 1990; Schwab, et al., 1989; Nishi,et al., 1988; Andreone, et al. 1989). These also are then interrupted byinsertion of an antibiotic resistance gene (e.g., puromycin) and clonedinto a targeting vector adjacent to a copy of, e.g., the herpes simplexvirus (HSV) thymidine kinase gene.

The plasmid is then introduced into cells by electroporation andhomologous recombination events are selected for by incubation of thecells in puromycin and FIAU, a recently described thymidine kinasesubstrate (Capecchi, 1989; available from Dr. Richard White, BristolMyers/Squibb, Walingford, Conn.). The action of FIAU is exerted asfollows. If recombination occurs at a nonhomologous site, the viralthymidine kinase gene is retained in the genome end expressed, renderingcells extremely sensitive to FIAU. If the disrupted gene is inserted atits homologous site (the endogenous hexokinase gene), in contrast, theviral thymidine kinase gene is lost, and the cells are tolerant of thedrug. While homologous recombination in mammalian cells is a relativelyrare event, the selection of strategy is sound, and has recently beenapplied to mammalian tissue culture cells (Zheng, et al., 1990).

Although glucokinase activity is present in AtT-20ins cells, theactivity of 0.7 U/g protein is only about 25% of the activity in normalislet cells, which contain approximately 3.1 U/g protein. One maytherefore desire to increase the glucokinase activity of engineeredcells, using a cDNA clone for the islet isoform of glucokinase (Newgard,1990; Hughes et al., 1991) and the strategies and vector systemsdescribed above. If the assumption about hexokinase overexpression iscorrect, it may be necessary to increase glucokinase expression inengineered cells having a reduced hexokinase activity in order toobserve any effects on glucose responsiveness. Note that creation of aGLUT-2⁺, insulin⁺, glucokinase-overexpressing, hexokinase- cell linewill require cotransfection of some of the relevant constructs, sincelimited numbers of resistance-gene containing plasmids are available.Efficient cotransfection can be expected when using eitherelectroporation or CaPO₄ precipitation transfection strategies(Sambrook, et al., 1989).

It is proposed that engineered cells that respond to glucose bysecreting insulin may be introduced into animals with insulin dependentdiabetes. Although ideally cells are engineered to achieve glucose doseresponsiveness more closely resembling that of islets, it is believedthat implantation of the CGT-5 or CGT-6 GLUT-2 expressing cells willalso achieve advantages in accordance with the invention. It should bepointed out that the experiments of Madsen and coworkers have shown thatimplantation of poorly differentiated rat insulinoma cells into animalsresults in a return to a more differentiated state, marked by enhancedinsulin secretion in response to metabolic fuels (Madsen, et al., 1938).These studies suggest that exposure of engineered cell lines to the invivo milieu may have some effects on their response(s) to secretagogues.

Engineered cells may be implanted using the alginate-polylysineencapsulation technique of O'Shea and Sun (1986), with modifications asrecently described by Fritschy, et al. (1991). The engineered cells aresuspended in 1.3% sodium alginate and encapsulated by extrusion of dropsof the cell/alginate suspension through a syringe into CaCl₂. Afterseveral washing steps, the droplets are suspended in polylysine andrewashed. The alginate within the capsules is then reliquified bysuspension in 1 ml- EGTA and then rewashed with Krebs balanced saltbuffer. Each capsule should contain several hundred cells and have adiameter of approximately 1 mm.

Implantation of encapsulated islets into animal models of diabetes bythe above method has been shown to significantly increase the period ofnormal glycemic control, by prolonging xenograft survival compared tounencapsulated islets (O'Shea, et al., 1986; Fritschy, et al., 1991).Also, encapsulation will prevent uncontrolled proliferation of clonalcells. Capsules containing cells are implanted (approximately1,000-10,000/animal) intraperitoneally and blood samples taken daily formonitoring of blood glucose and insulin.

Recently, further methods for implanting islet tissue into mammals havebeen described (Lacy et al., 1991; Sullivan et al., 1991; eachincorporated herein by reference). Firstly, Lacy and colleaguesencapsulated rat islets in hollow acrylic fibers and immobilized thesein alginate hydrogel. Following intraperitoneal transplantation of theencapsulated islets into diabetic mice, normoglycemia was reportedlyrestored. Similar results were also obtained using subcutaneous implantsthat had an appropriately constructed outer surface on the fibers. It istherefore contemplated that engineered cells of the present inventionmay also be straightforwardly "transplanted" into a mammal by similarsubcutaneous injection.

The development of a biohybrid perfused "artifical pancreas", whichencapsulates islet tissue in a selectively permeable membrane, has alsobeen reported (Sullivan et al., 1i9l). In these studies, a tubularsemi-permeable membrane was coiled inside a protecting housing toprovide a compartment for the islet cells. Each end of the membrane wasthen connected to an arterial polytetrafluoroethylene (PTFE) graft thatextended beyond the housing and joined the device to the vascular systemas an arteriovenous shunt. The implantation of such a device containingislet allografts into pancreatectomized dogs was reported to result inthe control of fasting glucose levels in 6/10 animals. Grafts of thistype encapsulating engineered cells could also be used in accordancewith the present invention.

An alternate approach to encapsulation is to simply inject glucosesensing cells into the scapular region or peritoneal cavity of diabeticmice or rats, where these cells are reported to form tumors (Sato, etal., 1962). Implantation by this approach may circumvent problems withviability or function, at least for the short term, that may beencountered with the encapsulation strategy. This approach will allowtesting of the function of the cells in experimental animals butobviously is not applicable as a strategy for treating human diabetes.

With what is learned from engineering of clonal cell lines, it mayultimately be possible to engineer primary cells isolated from patients.Dr. Richard Mulligan and his colleagues at the Massachusetts Instituteof Technology have pioneered the use of retrovirus vectors for thepurposes of introducing foreign genes into bone marrow cells (see, e.g,Cone, et al., 1984; Danos, et al., 1988). The cells of the bone marroware derived from a common progenitor, known as pluripotent stem cells,which give rise to a variety of blood borne cells includingerythrocytes, platelets, lymphocytes, macrophages, and granulocytes.Interestingly, some of these cells, particularly the macrophages, arecapable of secreting peptides such as tumor necrosis factor andinterleukin 1 in response to specific stimuli. There is also evidencethat these cells contain granules similar in structure to the secretorygranules of β-cells, although there is no clear evidence that suchgranules are collected and stored inside macrophages as they are inβ-cells (Stossel, 1987).

Nevertheless, it may ultimately be possible to use the recombinant DNAfor glucose transporters and glucose phosphorylating enzymes incombination with the recombinant insulin gene in a manner described forclonal cells to engineer primary cells that perform glucose-stimulatedinsulin secretion. This approach would completely circumvent the needfor encapsulation of cells, since the patient's own bone marrow cellswould be used for the engineering and then re-implanted. These cellswould then develop into their differentiated form (i.e., the macrophage)and circulate in the blood where they would be able to sense changes incirculating glucose by secreting insulin.

USE OF ENGINEERED CELLS FOR DIAGNOSIS OF IDDM PRIOR TO ONSET

As discussed above, antibodies against islet proteins have beenidentified in individuals with new-onset IDDM. The appearance of theseantibodies likely precedes the period of islet β-cell destruction andconsequent loss of insulin production. In recent years, significantprogress has been made in the identification of the specific proteinsthat are recognized by the immune system. Expression of one suchpotential antigen, the GLUT-2 islet β-cell glucose transporter, innon-islet cell lines, as described herein now allows us to test theimmune response of patient sera wit a specific islet antigen. Otherparticular epitopes contemplated by the inventor as being preferredinclude epitopes of cytoplasmic and surface islet cell antigens(Lernmark, 1982), insulin (Srikanta et al., 1986), proinsulin (Kuglin etal, 1988), islet 64 Kd and 38 Kd protein (Baekkeskov et al., 1982),immunoglobulins (DiMario et al., 1988), mammalian 65 Kd heat shockprotein (Elias et al., 1991), and even insulin receptors (Ludwig et al.,1987).

The inventors propose that cells engineered for specific expression ofone of the foregoing epitopes, or for any epitope that may subsequentlybe identified in autoimmune diabetes, may be employed in diagnostictests for diabetes. The principle of such a test involves reaction ofthe antibodies in a patients'serum with cells expressing the antigen(s)of choice, or epitope(s) of such an antigen, and subsequent detection ofthe antigen/antibody complex by reaction with a second antibody thatrecognizes human immunoglobulins (antibodies). A test would be scored aspositive if the serum being tested reacts with the cells engineered forexpression of the antigen of interest, but not with the parental(non-engineered) cell line. The reaction of the patient's serum with theexpressed antigen is measured indirectly by virtue of the fact that theanti-immunoglobulin antibody used is "labeled" or "tagged"with amolecule that allows its detection by direct inspection or mechanicalmeasurement. The most common "tags" that are linked to commerciallyavailable preparations of anti-human immunoglobulin are fluorescentmolecules such as fluorescein or tetramethyl rhodamine.

As disclosed hereinbelow, the use of engineered cells expressing theGLUT-2 antigen in diagnostic assays is greatly advantageous in that itallows rapid, efficient and reproducible analyses of patients'sera.Engineered GLUT-2-expressing cells, such as GLUT-2-expressingAtT20_(ins) cells may be used in diagnostic assays based either onimmunocomplex formation, or on the inhibition of glucose uptake, forexample, using 3-O-methyl-β-D-glucose. However, it will be understoodthat engineered cells expressing the GLUT-1 antigen will also haveutility. In particular, they may be used as `control` cells indiagnostic tests since no reaction of IDDM sera is detected withGLUT-1-expressing cells in these assays.

Regarding immunocomplex formation, two methodologies are available formeasuring the fluorescent signal resulting from formation of anantigen-antibody-anti-antibody complex. The first is simple directinspection of cells by fluorescence microscopy. In this procedure, cellsare adhered to poly-L-lysine coated microscope slides or cover slips.The cells are then fixed lightly by treatment with 0.5% paraformaldehydeor left untreated. Treatment of the cells with paraformaldehyde willcause changes in membrane structure of cells, resulting in changes inthe conformation of antigen molecules. For some, but not all antibodies,alteration of antigen conformation in thins way will allow a tighterassociation of the antibody and antigen. Engineered and control cellsare then exposed to either crude serum or purified immunoglobulins(IgGs) from patients to be tested for antibodies against the expressedantigen. After washing, the cells are exposed to an antibody recognizinghuman IgGs and the antigen/antibody/anti-antibody complexes arevisualized in a microscope by excitation of the fluorescent tag byexposure to light of an appropriate wavelength.

An alternative and more quantitative approach is to use a fluorescenceactivated cell sorter (FACS) to score immune complex formation. In thisprocedure, cells are treated with patient serum and labeled secondantibody much as described for the microscope slide approach except thatthe incubations are done with the cells in suspension rather thanattached to a slide. After treatment with the anti-human IgG antibody,cells are loaded into the FACS, which passes the cells one-by-one past alight source set at a wavelength that will excite the fluorescent markerof the second antibody. The cells then pass a detector which measuresthe fluorescence emission from the cells. Data are plotted as ahistogram of fluorescence intensity. A positiveantibody/antigen/anti-antibody reaction will result in an increase influorescence in most of the cells in a test. In contrast, exposure ofcells to sera that lack antibodies against the specific antigen beingpresented will result in little fluorescence. The utility of the FACS isthat it provides a display of the fluorescence intensity of all of thecells in a sample and plots the data as the distribution of fluorescenceintensities. Thus a positive sample will have a peak in celldistribution at a position on the graph that is shifted to the right(corresponding to a greater fluorescence intensity) relatively to asample that is not reactive.

To date the inventors have observed a noticeable increase in thefluorescent signal in GLUT-2 transfected AtT-20ins cells treated withsera from patients with IDDM compared with normal sera with both themicroscopic and FACS techniques. Importantly, an antibody raised againstan exposed (extracellular) region of the GLUT-2 molecule has been foundby the inventors to cause a shift (increase) in fluorescence that issimilar to the shift caused by the diabetic sera. Thus, GLUT-2 appearedto be a particularly useful epitope for the identification of new-onsetIDDM patients and even prediction of diabetes onset.

In copending application Ser. No. 483,224, filed Feb. 20, 1990 now U.S.Pat. No. 5,175,085, it is demonstrated that the sera of IDDM patientsincludes autoantibodies that are capable of inhibiting the uptake ofglucose by β-cells. This observation led to the development of abioassay for identifying individuals at risk for the development ofIDDM. Unfortunately, this method is somewhat cumbersome. Accordinglyvarious approaches were taken to simplify and improve this diagnosticassay, centering on the development of an immunological-based assay.

Among the approaches studied included ELISA- and Western blot-basedassays, as opposed to measurement of glucose transport rates. Attemptsat using these techniques were successful, but the problem at this levelwas the numbers of false positive normal individuals that wereidentified. Since there was a much better separation of the normal anddiabetic populations observed using the glucose transport assay, it washypothesized that the use of intact cellular protein in the transportassay, as opposed to the use of denatured protein in the Western blotand ELISA techniques, might account for the difference.

To test this hypothesis, artificial β-cells of the present inventionwere tested in the glucose transport assay. In these studies, it wasshown that IgG from IDDM patients effectively inhibited glucosetransport in the artificial β-cells, while no effect was seen with IgGsfrom normal individuals. Moreover, no effect of IgGs from new-onset Type1 individuals on glucose uptake was observed against cells that did notcontain the GLUT-2 protein.

These data led to the development of a flow cytometry-basedimmunofluorescence assay for antigen-antibody interaction between thepatient's autoantibodies and the glucose transporter mechanism. Initialattempts to develop such a system met with variable success. It wassuspected that this variability might be due to the day-to-day handlingof samples. Accordingly, a protocol was developed to ensure uniformgrowth of the cells, harvesting of the cells and treatment of the cellsunder conditions as close as possible to the transport assay. Theseconditions were as follows:

1. AtT 20 GT6 cells were grown for 72 hours following a 1:10 split atconfluence of the seed culture.

2. Cells were harvested from plates by scraping with a rubber policemaninto Dulbecco's phosphate-buffered saline at pH 7.6.

3. The cells were resuspended to a density of approximately 106cells/ml, washed by centrifugation at 500×g in Dulbecco'sphosphate-buffered saline, and incubated with shaking for 15 minutes at37° C. followed by 1 hour at 4° C. in 150 μl of patient serum.

4. Following two washes by centrifugation at 500×g in Dulbecco'sphosphate-buffered saline, the cells were resuspended in 200 μl ofR-phycoerythrin-labeled goat antihuman IgG (heavy chain specific),vortexed lightly, and incubated for 1 hour at 4° C. on a dual actionshaker.

5. Following two washes by centrifugation at 500×g in Dulbecco'sphosphate-buffered saline, the cells were resuspended in 500 μl ofDulbecco's phosphate-buffered saline and analyzed for antigen-antibodyinteraction using a flow cytometer.

As is discussed in greater detail in Example III, this flowcytometry-based immunofluorescence assay was found to be particularlyuseful in distinguishing the sera of patients with new-onset IDDM fromnon-diabetic subjects. It was found that 29 of 31 (94%) of thenondiabetic population were negative for IgG binding to GLUT-2 while 23of 30 (77%) of sera from IDDM patients were positive (FIG. 8). Thus, 81%of negative results were from nondiabetic patients and 92% of positiveresults were from patients with IDDM (Table 2).

USE OF ENGINEERED CELLS IN THE IDENTIFICATION OF SPECIFIC EPITOPES

The present inventors have recently discovered that GLUT-1 transfectedAtT-20ins cells do not discriminate diabetic from normal sera inFACS-based diagnostic tests, providing strong evidence that diabeticsera contain an antibody specific for the islet GLUT-2 glucosetransporter. It is therefore envisioned that the artificial β-cells ofthe present invention will be of use in the identification of thespecific epitope or segment of protein within GLUT-2 that is responsiblefor interacting with the antibody. Comparison of the GLUT-1 and GLUT-2sequences reveals that the 2 putative membrane spanning regions in thetwo molecules are highly hydrophobic and of very similar sequence. Thesehydrophobic segments are connected by "loops" of amino acids that havemuch less sequence conservation (see Bell, et al., 1990 for review). Inparticular, GLUT-2 contains a very large extracellular loop betweenmembrane spanning regions 1 and 2, while GLUT-1 contains a much smallerloop with little sequence homology to the GLUT-2 loop.

The inventors propose that construction of chimeric GLUT molecules inwhich individual or multiple "loop"regions are substituted could lead toidentification of the specific epitope of GLUT-2 that reacts withdiabetic sera. Thus, for example, the DNA encoding the largeextracellular loop of GLUT-2 can be inserted in place of the smallextracellular loop of GLUT-1 in the GLUT-1 cDNA sequence, and thischimeric molecule expressed in AtT-20ins cells. If the chimera reactswith diabetic serum (as-the native GLUT-1 molecule does not), the addedGLUT-2 extracellular loop would be the specific epitome. Once such anepitope is identified by the procedure outlined above, syntheticpeptides corresponding to this region of the protein sequence can beproduced and used to develop simpler diagnostic procedures. Exampleswould include a simple test in which the peptide epitope is reacted withtest serum and the formation of an antibody/peptide complex is monitoredby well established techniques such as ELISA or RIA.

INSULIN PRODUCTION FROM HIGH INSULIN-CONTENT ENGINEERED CELLS

GLUT-2 transfection is herein shown to cause an increase inintracellular insulin of approximately 5-fold in the AtT-20ins cellline, CGT-6. This finding demonstrates that batch extraction of insulindirectly from these or related cells is an alternative strategy forisolation and purification of human insulin for use in IDDM therapy.CGT-6 cells contain approximately 1 mUnit/10⁶ cells of human insulinwhen grown on gelatin beads in solution. The average IDDM patientrequires approximately 30 Units of insulin per day for control of bloodglucose levels. Cell densities of 5×10⁹ cells/liter cell culture mediaare readily achieved in the current liquid culture configuration,meaning that 5 Units of insulin/liter can be produced. Much higherdensities can be achieved using currently available commercialtechnology (e.g., that available from New Brunswick Scientific).

Furthermore, it is highly likely that the intracellular insulin contentof the cells can be further increased by one of the followingmethods: 1) Retransfection of AtT-20ins cells with the Rous sarcomavirus/human proinsulin of cDNA plasmid that contains a resistance genesuch as the neomycin resistance gene. The level of expression of atransfected gene appears to be dependent on the site of insertion of theplasmid in the chromosome. Thus, it is highly likely that higher levelsof insulin expression will be achieved by simply reintroducing theplasmid and isolating new resistant clones. 2) Construction of plasmidsin which human proinsulin cDNA expression is directed by alternatepromoters. Examples include the CMV promoter, which was used to achievevery high levels of expression of GLUT-2 in the creation of the CGT-6cell line in the inventor's laboratory, or 3) Amplification of the viralpromoter/human proinsulin cDNA (Sambrook, et al., 1989) by cloning nextto a resistance gene such as dihydrofolate reductase (DHFR), adenosinedeaminase, or glutamine synthetase (Cockett, et al., 1990). Of these,DHFR is the most commonly used system, but is generally of limitedusefulness in cell lines that have endogenous expression of DHFR (thisis true of the AtT-20ins cell line). The glutamine synthetase systemallows amplification of the gene of interest even in the presence ofendogenous expression of glutamine synthetase.

Cells are stably transfected with a plasmid containing the transcriptionunit (i.e., viral promoter hooked to the human proinsulin gene) adjacentto the hamster glutamine synthetase coding sequences. Selection ofclones and amplification of the integrated transcription unit/GS gene isthen carried out by addition of methionine sulfoxide to the tissueculture media (Cockett, et al., 1990). Resulting clones contain greatlyincreased copy numbers of the transcription unit, by virtue of itsassociation with the amplified glutamine synthetase gene. As a result,much greater quantities of insulin are produced by the recombinant cell,making it an even more viable source for human insulin production.

LIQUID CULTURE OF ENGINEERED CELLS FOR INSULIN PRODUCTION

As there has been little progress in developing new strategies fortreating diabetes, therapy for diabetic patients is still centeredaround repeated self-injections of insulin. The methods employed for theproduction of human insulin to be used in this manner currently includeeither chemically complexing purified recombinant insulin A and Bchains, or purifying pig insulin from freshly isolated porcine pancreasor pancreatic islets. Both of these methods are technically difficultand laborious, and the latter is additionally complicated by thepresence of many active proteases in the tissue of origin.

In considering the drawbacks of the methods currently employed forinsulin production, the invention contemplates that correctly-foldedhuman insulin could be produced relatively simply and rapidly usingclonal βcells that secrete insulin in response to glucose.

The most appropriate method to accomplish this has been found by theinventors to be the perifusion of a column containing CTG-6 cellsadhered to gelatin beads. Passing a glucose-containing buffer, such asKRS, 5 mM glucose, pH 7.4, over such a column of such artificial β cellshas been found to stimulate the increased secretion of insulin into thesurrounding media, which can then be collected and used as a startingmaterial for the purification of recombinant insulin.

It is anticipated that purification of insulin from the perifusion mediacan be rapidly achieved by one or a combination of the followingapproaches: 1) Affinity chromatography, for example, passage of theinsulin containing media over a column containing anti-insulinantibodies. After removal of non-insulin proteins and other impuritiesby washing of the column, insulin can be specifically eluted by using abuffer with an increased salt concentration or decreased pH. 2)Preparative high performance liquid chromatography. 3) Size selection byconventional size-exclusion column chromatography.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representlaboratory techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE I ENGINEERING OF GLUCOSE-STIMULATED INSULIN SECRETION INNON-ISLET CELLS

A. Methods

1. AtT-20ins cell culture and tissue isolation

The AtT-20ins cells used were provided by Dr. Regis Kelly, University ofCalifornia San Francisco, and were similar to the line that wasoriginally described (Moore, et al., 1983) except that the Rous sarcomavirus long terminal repeat was substituted for the SV40 early genepromoter for directing insulin cDNA expression. The cells were grown inDulbecco's modified Eagles' medium (DMEM), supplemented with 10% fetalcalf serum, 100 μg/ml streptomycin, and 250 μg/ml neomycin. Anteriorpituitary and liver samples were excised from normal ad-lib fed Wistarrats, and islets were isolated from groups of 10-20 animals aspreviously described (Johnson, et al., 1990a, 1990c) and pooled for RNAextraction or homogenization for glucose phosphorylation assays.

2. Stable transfection of AtT-20ins cells with GLUT-2

The rat islet GLUT-2 cDNA (Johnson, et al., 1990s) was cloned into thevector pCB-7, a derivative of vector pCMV4 (Anderson, et al., 1989),immediately downstream of its cytomegalovirus (CMV) promoter. The cDNAwas cleaved at its 3' end with Hind III, resulting in the removal of 635base pairs of 3' untranslated region. AtT-20ins cells were transfectedwith this construct using electroporation. Cells were harvested frompre-confluent plates by light trypsinization, washed twice in phosphatebuffered saline, and resuspended at 3×10⁶ cells/ml in a solutioncontaining 20 mM Hepes (pH 7.05), 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HP)₄, 6 mM glucose, and 0.5 mg/ml salmon testis DNA. After equilibrationof the cells to room temperature in electroporation cuvettes (Bio-RadLabs; electrode gap width 0.4 cm), a single pulse was delivered using acapacitance setting of 960 μF and voltage settings between 0.2 and 0.3kV. The cells remained in the buffer for five minutes and were thenplated onto tissue culture dishes. Stable transfectants were selectedwith hygromycin, since the plasmid also contains a resistance gene forthis drug. Four colonies were obtained and passaged several times in thepresence of hygromycin to obtain a pure stock.

3. RNA blot hybridization analysis

RNA was prepared by guanidinium isothiocyanate extraction, resolved on aformaldehyde/agarose gel and transferred to a nylon membrane (MicronSeparations Inc.) as previously described (Newgard, et al., 1986). Blotswere hybridized sequentially with 32p labeled antisense GLUT-2 or 18SrRNA probes, prepared as described (Chen, U et al., 1990), withstripping of the blot between hybridizations by boiling in 0.1% SDS for30 minutes.

4. Immunoblot analysis

Liver plasma membranes were prepared by the method of Axelrod and Pilch(1983) and only the light plasma membrane fraction was used. Islet andAtT-20ins cell membranes were prepared as previously described (Johnson,1990b), except that the sucrose gradient was deleted and thehomogenization buffer consisted of 50 mM Tris, pH 7.4, 5 mM EDTA, 0.1 mMp-chloromercurlbenzene sulfonate (PMSF), 10 mM benzamidine, and 1tTrasylol. The samples were transferred onto nitrocellulose andimmunoblotted exactly as described (Hughes, et al., 1991; Quaade, etal., 1991), either with a 1:1000 dilution of the anti GLUT-2 polyclonalantiserum (Johnson, et al., 1990b), or with the diluted antiserum after10 minutes of preincubation with an equal volume of a 1 mg/ml solutionof the antigenic peptide dissolved in PBS. The second antibody was ¹²⁵I-labeled goat anti-rabbit anti-IgG, and the resultant immune complexeswere visualized by autoradiography.

5. GLUT-2 immunofluoresconce in AtT-20ins cells

Parental AtT-20ins cells or transfected lines CGT-5 and CGT-6 were grownto a density of 5×10⁶ cells per 100 mm dish and harvested by incubationat 30° C. with a solution of 0.02% EDTA in PBS. After three washes inDMEM containing 20 mM HEPES, approximately 1.5×10⁵ cells weretransferred onto 12 mm poly-L-lysine coated glass coverslips, to whichthey adhered during a 30 minute incubation at 37° C. The cells were thenfixed with 3% paraformaldehyde in PBS for 30 minutes at roomtemperature, and incubated with 0.1M NH₄ Cl in phosphate buffered saline(PBS), pH 7.9 for 30 minutes. After 4 rinses with PBS, cells werepermeabilized with 0.1% TRITON™ X-100 for 5 minutes, then rinsed 3 timeswith PBS. After a pre-incubation with 2% BSA, GLUT-2 antiserum (1:2500)was applied in the presence or absence of an equal volume of theantigenic peptide (1 mg/ml). Slides were incubated overnight, and excessantibody removed by washing 5 times with 0.1% BSA in 0.1M phosphatebuffer, pH 7.9. Cells were then incubated with FITC-conjugated goatanti-rabbit IgG for two hours at 37° C. and washed sequentially withBSA/phosphate buffer and water. After application of coverslips, theslides were visualized by fluorescent light microscopy.

6. Glucose transport measurements

Cells were harvested by scraping with a rubber policeman, washed inHanks balanced salt solution by centrifugation at 600×g, and resuspendedin Dulbecco's modified Eagles's media with 10% fetal calf serum and 5 mMglucose. Cells were incubated at 37° C. for 30 minutes, washed,resuspended in phosphate buffered saline and assayed for 3-O-methylglucose uptake as previously described (Johnson, et al., 1990c). Resultswere expressed as mmoles 3-O-methyl glucose uptake/min/liter cell space.Initial velocities of uptake were derived from duplicated measurementsat 3, 6, and 15 seconds for each concentration of glucose with thetransfected cell lines and 3, 15, and 30 seconds for the parental cellline (due to slower transport rate in these cells).

7. Glucose phosphorylation assays

Glucose phosphorylation and glucokinase activities were measured byconversion of U-¹⁴ C glucose to U-¹⁴ C glucose-6-phosphate, aspreviously described (Method "B"in Kuwajima, et al., 1986). Culturedcells or tissues were homogenized in 5 volumes of buffer containing 10mM Tris, 1 mM EDTA, 1 mM MgCl₂, 150 mM KCl, and 1 mM DTT, pH 7.2. Thehomogenate was cleared by centrifugation at 12,000×g and the supernatantused for assays of glucose phosphorylation. Reactions were carried outat 37° C. in a total volume of 150 μl, and initiated by addition of10-30 μl of extract to a reaction mix containing 100 mM Tris, 5 mM ATP,10 mM MgCl₂, 100 mM KCl, 1 mM DTT, pH 7.2, 15 or 50 mM glucose, and 6.2μCi of U-¹⁴ C glucose (300 mCi/mmol; New England Nuclear). In order todiscriminate glucokinase and hexokinase activities, assays wereperformed in the presence and absence of 10 mM glucose-6-phosphate,which potently inhibits hexokinase but not glucokinase activity.Reactions were carried out for 90 minutes and terminated by addition of50 μl of reaction mix to 100 μl of 3% methanol in 95% ethanol. Analiquot of this mixture was transferred to nitrocellulose filter circles(Grade NA 45, Schleicher & Schuell), which bind phosphosugars, and afterair drying, washed extensively in water to remove labeled glucose.Radioactivity on the paper was then detected by liquid scintillationcounting, and glucose phosphorylating activities are expressed in termsof the total protein content of the extracts.

8. Insulin secretion from AtT-20ins cells in response to secretagogues

Parental or GLUT-2 transfected lines CGT-5 and CGT-6 were removed fromgrowth plates by light trypsinization and replanted in 6 well dishes(Costar) at a density of 5×10⁵ cells per well. The cells were then grownfor three days in culture media containing 1 mM (see above). On thethird day, cells were washed twice for 10 minutes each in HEPES balancedsalt solution containing 1% BSA (HBSS), but lacking glucose. Secretionexperiments were initiated by addition of HBSS plus a range of glucoseconcentrations (0-20 mM) or in the presence of one of three non-glucosesecretagogues, forskolin (0.5 μM), dibutyryl cAMP (5 mM), orisobutylmethylxanthine (IBMX, 0.1 mM), in the presence or absence ofglucose. Cells were incubated with secretagogues for 3 hours, afterwhich media was collected for insulin radioimmunoassay.

9. Assay of intracellular insulin

Cells were collected in 1 ml of 5M acetic acid, lysed by three cycles offreeze-thawing, and lyophilized. The dried lysate was then reconstitutedin 5 ml of insulin assay buffer (50 mM NaH₂ PO₄, 0.1% BSA, 0.25% EDTA,1% aprotinin, pH 7.1) and aliquots were assayed for insulin byradioimmunoassay.

B. Results

1. Expression of GLUT-2 mRNA in transfected AtT-20ins cells

Expression of GLUT-2 mRNA was evaluated by blot hybridization analysisof AtT-20ins cells, either transfected or untransfected with acytomegalovirus (CMV) promoter/GLUT-2 hybrid gene, and in extracts ofrat liver, islets of Langerhans, and anterior pituitary tissues. Theradiolabeled GLUT-2 antisense RNA probe (Johnson, et al., 1990a; Chen,et al., 1990) was hybridized to a blot containing equal amounts of RNAfrom four GLUT-2 transfected AtT-20ins cell lines (CGT-1, CGT-2, CGT-5,CGT-6), untransfected AtT-20ins cells, and the three primary tissues(FIG. 1). Steady state levels of GLUT-2 mRNA were highest in CGT-5 andCGT-6; the former contained approximately half as much and the latter anequal amount of GLUT-2 mRNA as rat islets, and they contained 10 and 16times as much, respectively, as rat liver, measured by densitometricscanning and normalization to the signal obtained with an 18SrRNA probe.The transfected lines contained a smaller GLUT-2 transcript than liveror islets (2.2 versus 2.8 kb) because 635 base pairs of the 3'untranslated region were removed in the course of cloning the GLUT-2cDNA into the pCB-7 vector. Lines CGT-1 and CGT-2 exhibited less activeexpression of GLUT-2. Untransfected AtT-20ins cells and primary anteriorpituitary cells did not contain detectable amounts of GLUT-2 mRNA,consistent with previous studies (Hughes, et al., 1991).

2. Expression of GLUT-2 protein in tissues and cell lines

In order to evaluate the levels and molecular status of the expressedGLUT-2 protein in transfected AtT-20ins cells, we resolved crudemembrane fractions by SDS/PAGE, transferred the proteins tonitrocellulose, and detected GLUT-2 protein with an antibody raisedagainst its C-terminal hexadecapeptide sequence (Johnson, et al.,1990b). The antibody recognized two distinct bands in liver and islets,with apparent molecular weights of 70 and 52 kd in liver and slightlydifferent sizes of 72 and 56 kd in islets (FIG. 2, left). Consistentwith the RNA blot hybridization data, untransfected AtT-20ins cells werefound to lack GLUT-2 protein, while a single intense band ofapproximately 70 kd was observed in extracts from either of thetransfected lines CGT-5 and CGT-6. The specificity of the antibody isdemonstrated by the fact that all bands were blocked by preincubation ofthe antibody with the antigenic peptide (FIG. 2, right). Thorens, et al.(1988) have previously reported that a similar anti-peptide antibodyrecognizes GLUT-2 proteins of distinct molecular weights in liver (53kd) and islets (55 kd), despite the fact that the cDNA sequences forGLUT-2 are identical in liver and islets in both rat (Johnson, et al.,1990a) and man (Permutt, et al., 1989). They did not report on thelarger bands shown herein, possibly because of differences in theprotocols used for membrane preparation.

3. Immunecytochemistry of GLUT-2 in transfected AtT-20ins cells

Expression of GLUT-2 protein in transfected AtT-20ins cells was studiedby immunofluorescent staining techniques, using an antibody raisedagainst the C-terminal hexadecapeptide of GLUT-2 (Johnson, et al.,1990b). In the lines with highest GLUT-2 mRNA levels (CGT-5 and CGT-6),abundant GLUT-2 immunofluorescence was detected at the cell membrane aswell as some intracellular signal that was mostly polarized to regionsof cell-cell contact. The signal was blocked by preincubation of theantibody with the antigenic peptide and was not seen in untransfectedcells or in cells transfected with the vector lacking the GLUT-2 insert.Expression of GLUT-2 protein in transfected AtT-20ins cells and itsabsence in the untransfected parental line was confirmed by immunoblotanalysis. Thus, AtT-20ins cells and its absence in the untransfectedparental line was confirmed by immunoblot analysis. Thus, AtT-20inscells not only have the capacity to produce GLUT-2 mRNA and protein butalso sort the protein to the cell membrane, as occurs in both islets andliver (Thorens, et al., 1988; Orci, et al., 1989; Tal, et al., 1990;Orci, et al., 1990). Preferential expression at regions of cell-cellcontact is in keeping with a recent report (Orci, et al., 1989) showingthat GLUT-2 expression in islet β-cells is not homogenous and is mostabundant in regions of membrane enriched in microvilli and facingadjacent endocrine cells, as opposed to regions facing capillaries orempty spaces between cells. The functional significance of thisphenomenon is currently not understood.

4. Glucose transport measurements in parental and GLUT-2 expressionAtT-20ins cells

The GLUT-2 cDNA has been cloned from both liver (Thorens, et al., 1988)and islets (Permutt, et al., 1989; Johnson, et al., 1990a), two tissueswith high Km glucose transport activity. Although the cDNA has beenexpressed in bacteria (Thorens, et al., 1988) and oocytes (Permutt, etal., 1989), these systems have not been used for kinetic studies. Thus,direct evidence that the GLUT-2 cD.A encodes a protein that confers thehigh Km glucose transport activity has not been presented to date.

Dramatic differences in glucose transport kinetics were found betweentransfected and untransfected AtT-20ins cells. FIG. 3A shows a plot ofthe concentration dependence of glucose uptake in the AtT-20ins celllines, and demonstrates the dramatically increased rates of glucosetransport in lines CGT-5 and CGT-6 relative to the untransfected(parental) AtT-20ins cells. Lineweaver-Burke analysis of the data showedthat the CGT-5 and CGT-6 lines had apparent Kms for glucose of 16 and 17mM and Vmax values of 25 and 17 mmoles/min/liter cell space,respectively (FIG. 3B). In contrast, the untransfected parentalAtT-20ins line had an apparent Km for glucose of 2 mM and a Vmax of 0.5mmoles/min/liter cell space (FIG. 3C), consistent with its expression ofthe GLUT-1 mRNA (Hughes, et al., 1991), which encodes the low Km glucosetransporter found in most clonal cell lines (Flier, et al., 1987;Birnbaum, et al., 1987). The transfected AtT-20ins cells have glucosetransport kinetics that are remarkably similar to isolated, dispersedislets of Langerhans, which have a Km of 18 mM for glucose and a Vmax of24 mmoles/min/liter cell space (Johnson, et al., 1990a). Thus, theGLUT-2 cDNA clearly encodes the protein responsible for the high Kmglucose transport activity in islets and liver, and is capable oftransferring this activity into the AtT-20ins cell line.

5. Glucose-stimulated insulin secretion from AtT-20ins cells

Insulin secretion from GLUT-2 transfected and untransfected cells wasmeasured over a range of glucose concentrations from 0-20 mM. FIG. 4Acompares glucose-stimulated insulin release from AtT-20ins cells andCGT-6 cells, expressed as mU insulin released/mg total cellular protein.Consistent with previous results (Hughes, et al., 1991), glucose had nosignificant effect on insulin release from parental AtT-20ins cells.AtT-20ins cells transfected with the pCB7 vector lacking a GLUT-2 insertwere also found to be unresponsive to glucose. GLUT-2 transfected cells,in contrast, are clearly glucose responsive (data are shown for lineCGT-6 only; results for line CGT-5 were qualitatively identical). Asubmaximal but statistically significant (p=0.002) increase in insulinrelease relative to insulin release at 0 mM glucose was observed at thelowest concentration of glucose studied (5 μM); maximal stimulation ofapproximately 2.5-fold was observed at all higher concentrations overthe range 10 μM-20 mM (p≦0.001). It is highly unlikely that theseresults can be attributed to clonal selection of glucose responsivesubpopulations of the parental AtT-20ins cells, since cells transfectedwith vector lacking GLUT-2 failed to respond, while two independentGLUT-2 expressing lines (CGT-5 and CGT-6) gained glucose sensing.

In normal islets, glucose potentiates the insulin secretory response tovarious β-cell secretagogues, including agents that increaseintracellular cAMP levels (Ullrich and Wollheim, 1984; Malaisse, et al.,1984). The potentiating effect of glucose on insulin secretion in thepresence of forskolin, dibutyryl cAMP, and IBMX was therefore studied.Glucose had a modest stimulatory effect on forskolin stimulated insulinrelease from parental AtT-20ins cells, expressing the data either asinsulin release/mg cellular protein (FIG. 4A) or as insulin release/mgcellular DNA (FIG. 4B). In contrast, glucose had a powerful potentiatingeffect on forskolin stimulated insulin release from transfected CGT-6cells. The response was unchanged by glucose concentration over therange of 1-5 mM, and similar potentiating effects of glucose on dibutrylcAMP and IBMX induced secretion were also observed.

Insulin secretion experiments involved static incubation of cells withthe secretagogue for three hours, and thus provided little informationabout the dynamics of insulin release. The inventors succeeded ingrowing the parental and transfected AtT-20ins cell lines on gelatinbeads in liquid culture, thus allowing their secretory properties to bestudies by perfusion with glucose containing media. As shown in FIG. 9,cells grown in this configuration released insulin within minutes ofglucose stimulation. Furthermore, the insulin secretory responseexhibits a first intense and a second less intense but sustained phase,as is characteristic of normal β cells.

6. Insulin Content of Native and Engineered AtT-20ins cells

A remarkable finding of this study is that transfection of AtT-20inscells with the GLUT-2 cDNA results in a substantial increase inintracellular insulin content, despite the fact that insulin geneexpression is driven by the glucose insensitive Rous sarcoma viruslong-terminal repeat enhancer/promoter in these cells. Native AtT-20inscells and the GLUT-2 transfected CGT-6 cells were grown for 3 days inmedia supplemented with low (1 mM) or high (25 mM) glucose. The CGT-6cells were found to contain 3.6-fold and 5.4-fold more insulin than theAtT-20ins cells when studied at low and high glucose, respectively(p<0.001 for both comparisons). Furthermore, insulin content wasapproximately double in the CGT-6 cells grown at high glucose comparedwith the same calls grown at low glucose (p<0.001). In contrast, in theuntransfected AtT-20ins cells, high glucose caused only a 20% increasein insulin content.

7. Glucose phosphorylation in AtT-20ins cells

AtT-20ins cells were transfected with GLUT-2 secrete insulin at glucoseconcentrations that are substimulatory for islets. The enhancedsensitivity to glucose is not explained by the kinetics of glucosetransport, since both the CGT-5 and CGT-6 lines transport glucose with avelocity and concentration dependence that is virtually identical toislets. Alternatively, stimulation of insulin secretion at low glucoseconcentrations might be explained by differential regulation of glucosephosphorylation in AtT-20ins cells relative to β-cells. The ratio ofhexokinase-glucokinase activity in these cells was therefore comparedwith activities found in normal islets of Langerhans and liver. Studiesfrom this and other laboratories (Iynedjiian, et al., 1989; Magnuson andShelton, 1989; Newgard, et al., 1990; Hughes, et al., 1991) have shownthat the single glucokinase gene is alternatively regulated andprocessed in liver and islets, resulting in distinct transcripts thatpredict proteins with unique N-termini; the Km for glucose of bothisoforms is in the range of 8-10 mM. AtT-20ins cells express the isletisoform of glucokinase (Hughes, et al., 1991).

A radioisotopic glucose phosphorylation assay was performed (Method "B"in Kuwajima, et al., 1986) that allows discrimination of glucokinase andhexokinase activities when performed in the presence and absence of 10mM glucose-6-phosphate, since this metabolite is a potent inhibitor ofhexokinase, but not glucokinase (Wilson, 1984). As shown in Table 1,total glucose phosphorylating capacity and glucokinase activity are notsignificantly different in transfected (line CGT-6) versus untransfected(parental) AtT-20ins cells. Both lines have a total glucosephosphorylating capacity that is similar to that in liver and islets.However, glucokinase activity in AtT-20ins cells is only 32% of theglucokinase activity in islets and 10% of that in liver. Moreover,glucokinase represents only 9% of the total glucose phosphorylatingactivity of AtT-20ins cells (the remaining 91% is presumably due tohexokinase activity), as compared to 24% in normal islets and 86% innormal liver. The altered hexokinase:glucokinase ratio in AtT-20inscells may result in low Km glucose metabolism that accounts for theinsulin secretory response at low glucose concentrations.

                  TABLE 1                                                         ______________________________________                                        Glucose Phosphorylating Activities in Tissues                                 and Cell Lines.                                                                        Total Glucose*                                                                             Glucokinase#                                                                             Glucokinase                                           Phosphorylation                                                                            (U/gram    (% of                                        Cell Type                                                                              (U/gram protein)                                                                           protein)   total)                                       ______________________________________                                        AtT-20ins.                                                                             9.19 ± 0.27                                                                             0.63 ± 0.06.sup.a                                                                     6.8%                                         (parental)            0.43 ± 0.08.sup.b                                    AtT-20ins                                                                              8.09 ± 0.20                                                                             0.86 ± 0.18.sup.a                                                                     10.6%                                        (line CGT-                                                                    6)                                                                            Islet    9.61 ± 2.10                                                                             2.31 ± 0.35.sup.a                                                                     24.0%                                        Live     8.42 ± 1.09                                                                             7.19 ± 1.31.sup.a                                                                     85.4%                                        ______________________________________                                         *Total glucosephosphorylation was measured in 14,000 × g supernatan     of crude homogenates, at 50 mM glucose, using an assay that monitors          .sup.14 C glucose conversion to .sup.14 C glucose6-phosphate ("Method B"      in Kuwajima, et al., 1986). #Glucokinase activity was determined with the     same assay as used for total glucose phosphorylation at 50 (.sup.a) or 15     (.sup.b) mM glucose, except in the presence of 10 mM glucose6-phosphate t     inhibit hexokinase. Values represent the means ± SEM for 3 independent     determinations for liver and islets and 4 independent determinations for      untransfected (parental) and GLUT2 transfected (line CGT6): AtT20ins          cells.                                                                   

EXAMPLE II DIAGNOSIS OF IDDM

A. Methods

1. Direct inspection of immunoreactive cells by fluorescence microscopy

Parental and engineered AtT-20ins cells are grown to a density of 5×10⁶per 100 mm dish and harvested by incubation at 37° C. with a solution of0.02% EDTA in phosphate buffered saline (PBS). After washing the cellsin DMEM media containing 20 mM Hepes, approximately 1.5×10⁵ cells aretransferred onto 12 mm poly-L-lysine coated glass coverslips, to whichthey adhere during a 30 minute incubation at 37° C. The cells are thenfixed for 30 minutes with varying amounts (0.5-3.0%) ofparaformaldehyde, depending on the extent of fixation that is desired.For studies with anti-GLUT-2 antibodies or serum, the inventors havefound a light fixation (0.5% paraformaldehyde) to be most appropriate.After preincubation with 2% BSA, a serum sample (usually diluted 1:1 inBSA) is added to the sample in sufficient volume to cover the cells. Asa positive control, an antibody (designated X617) raised against theunique extracellular loop peptide of the rat GLUT-2 transporter is used,diluted 1:100 in PBS (the antibody is raised against a peptide withsequence DAWEEETEGSAHIV (SEG ID NO. 1), as found at amino acids 64-77 ofthe rat GLUT-2 primary structure).

Slides are incubated overnight with serum or antibody, and excessantibody is removed by washing with 0.1% BSA in 0.1M phosphate buffer,pH 7.9. Cells are then incubated with FITC-conjugated goat anti-humanIgG (in the case of human serum samples) or FITC-conjugated goatanti-rabbit IgG (in the case of antibody X617, which was raised inrabbits). After application of coverslips, the slides are visualized byfluorescent light microscopy. A test is scored as positive if for aparticular serum sample, a clear fluorescent signal is seen at themembrane surface of GLUT-2 expressing AtT-20ins cells but not inparental AtT-20ins cells. A positive response with antibody X617 furtherproves that the CLUT-2 protein is expressed in proper orientation andthat epitopes that are expected to reside at the cell surface are indeedrecognizable.

2. Use of a fluorescence activated cell sorter (FACS) to score immunecomplex formation

Cells are prepared for FACS analysis essentially as described for themicroscope slide approach except that incubations are done with cells insuspension rather than attached to microscope slides. Briefly,near-confluent tissue culture plates containing parental AtT-20ins cellsor GLUT-2 expressing CGT-6 cells are washed with PBS, and then exposedto 0.02% EDTA for 15 minutes at 37° C. to dislodge cells from the plate.The dispersed cells are washed with culture media followed by PBS andused as intact, live cells or fixed gently in 0.5% paraformaldehyde/PBSfor 15 minutes at room temperature. The live or fixed cells are thenincubated in 100 μl of patient serum: PBS in a ratio of 1:1 with 0.002%EDTA added to keep the cells dispersed. After a one hour incubation at4° C., the cells are washed 3 times with PBS and incubated withanti-human IgG or anti-human globulin fraction labeled withphycoerythrin for 1 hour at 4° C. Subsequently, the cells are washedwith PBS and run through a flow cytometer in the red channel.Phycoerythrin is chosen as the fluorescent marker because we found theAtT-20ins cells have a natural fluorescence in the green channel that isused for FITC-labeled antibodies.

B. Results

1. Microscope slide technique

Use of the antibody raised against the external loop peptide of GLUT-2in the inventor's laboratory (X617) results in a clear fluorescentstaining at the surface of engineered AtT-20ins cells that expressGLUT-2, but gives no such signal in parental cells that have not beenengineered for GLUT-2 expression. Furthermore, the signal in GLUT-2transfected cells can be blocked by preincubation of antibody X617 withthe peptide to which it was raised. These results indicate thatformation of an immune complex with an external (extracellular) epitopeof the GLUT-2 protein can occur and is readily detectable. Inpreliminary studies with sera isolated from new-onset Type I diabeticpatients (ranging in age from 10-20 years old), and age matched normalcontrols, the diabetic sera, but not the normal sera show a greaterimmunoreactivity against the GLUT-2 transfected cells relative to theuntransfected controls.

2. FACS technique

The FACS method was found to be appropriate for detecting the presenceof a specific immune complex (FIG. 5A and FIG. 5B). Graphs 1 and 2 ofFIG. 5A were derived by treatment of GLUT-2 expressing AtT-20ins cellswith the anti-GLUT-2 antibody X617 and treatment with anti-rabbit IgGsecond antibody labeled with phycoerythrin. Graphs 3 and 4 representcells incubated with antibody X617 after it had been preincubated withGLUT-2 expressing AtT-20ins cells. The cells are loaded into the FACS,which passes the cells one-by-one past a light source set at awavelength that will excite the fluorescent marker of the secondantibody. The cells then pass a detector which measures the fluorescenceemission from the cells. Data are plotted as a histogram of fluorescenceintensity. As can be seen, curves 1 and 2 are shifted to the rightrelative to curves 3 and 4, indicating a greater fluorescence intensityin those cells. A similar experiment was performed with parentalAtT-20ins cells not expressing GLUT-2 (FIG. 5B). In these cells, nodifference is seen between the naked antibody and antibody preabsorbedwith GLUT-2 expressing cells. Taken together, these data serve tovalidate the technique, in that a specific response can be measured toan antibody known to react with an extracellular-domain of GLUT-2.

This method was used in the preliminary analysis of serum from adiabetic patient (FIG. 6A, FIG. 6B and FIG. 6C). FIG. 6A shows thefluorescence spectrum of GLUT-2 transfected AtT-20ins cells incubatedwith the second antibody (phycoerythrin labeled anti-human globulin)alone. In FIG. 6B, the GLUT-2 transfected cells have been incubated withserum isolated from a normal patient, resulting in a shift in thefluorescence intensity relative to the control in FIG. 6A. In FIG. 6C,cells are incubated with serum from a patient with new-onset Type Idiabetes. Importantly, this serum causes a much more pronouncedrightward shift in fluorescence relative to the normal or nonserumcontrols. The sample shown is representative of most other diabetic andnormal sera assayed to date.

EXAMPLE III INTERACTIONS OF SERA FROM DIABETIC PATIENTS WITH ISLET CELLSAND ENGINEERED AtT20_(ins) CELLS

The following example is directed to an analysis of serum samples fromdiabetic patients and non-diabetic subjects. In particular, theinteractions of purified IgG samples with rat islet cells and engineeredAtT20_(ins) cells was investigated using both binding assays and assaysbased on the inhibition of glucose uptake. The following resultsdemonstrate the usefulness of such analyses in diagnostic and prognostictests.

A. Immunofluorescence/Flow Cytometric Methods

AtT20_(ins) cells and GLUT-2-expressing AtT20_(ins) cells were harvestedby removal of cells from plates with a rubber policeman in Dulbecco'sphosphate-buffered saline, pH 7.6. Following two washes in Dulbeccolsphosphate-buffered saline by sedimentation at 500×g for 30 seconds atroom temperature, the cells were divided into 1.5 ml microfuge tubes ata density of approximately 10⁵ cells per tube. Cells were incubated for1 hour at 4° C. in 150 μg of patient sera with occasional agitation. Thecells were then washed twice by centrifugation at 500×g for 30 secondsin Dulbecco's phosphate-buffered saline pH 7.6 and resuspended inR-phycoerythrin-labeled goat antihuman, heavy chain-specific IgGCR-PEAb) (Fisher Scientific) and incubated for 1 hour at 4° C. withoccasional shaking. Following an additional two washes by centrifugationat 500×g for 30 seconds in Dulbecco's phosphate-buffered saline, pH 7.6,the cells were resuspended in 500 μl of Dulbecco's phosphate-bufferedsaline, pH 7.6, and analyzed for IgG binding using flow cytometry.

Flow cytometry was performed on a FACScan (Becton Dickinson) flowcytometer. Forward scatter threshold was set at 100 using the E-01forward scatter detector. Linear amplifier gains were 6.18 for forwardscatter and 1.22 for 90° angle light scatter with a photomultipliersetting of 274 volts. Forward and 90° angle light scatter were read onlinear scale and fluorescence measurements were made on logarithmicscale. Setting adjustments were made by using a sample of unstainedcells and increasing the photomultiplier voltage so that events wereon-scale during observation of 530±15 nm (FL1) histogram. A sample ofcells stained only with R-phycoerythrin-labeled goat antihuman IgG(R-PEAb) was then used to adjust the photomultiplier voltage so thatevents were on-scale during measurement of a 575±13 nm (FL2) histogram.A control specimen was then used to adjust the FL2 photomultiplier tubevoltage such that FL2 histogram events remain minimally on scale. TheFL2-FL1 compensation was adjusted to minimize fluorescence overlap andfor these cells a setting of 45.9% was used. Acquisition of 10⁴ eventsper specimen were required and data were stored on floppy discs foranalysis.

B. Results

1. Effects of IgG from Diabetic Patients and Nondiabetic Subjects on3-O-Methyl-β-D-Glucose Uptake by Islet Cells and GLUT-2-Expressing andGLUT-1-Expressing AtT20_(ins) Cells

The following assays were performed to investigate the effects of humanIgG on glucose uptake by intact cells. The assays were performed asdescribed by Johnson and Unger, PCT Patent Application WO 91/13361,incorporated herein by reference.

Examination of the effects of purified IgG from 7 patients withnew-onset IDDM and 6 nondiabetic individuals revealed that3-O-methyl-β-D-glucose uptake by rat islet cells was significantlyinhibited in the presence of IgG from patients with IDDM (FIG. 7A, FIG.7B and FIG. 7C). Initial rates of uptake averaged 15 mmoles3-O-methyl-glucose/min/liter islet cell space in the presence of IgGfrom nondiabetic patient sera versus 9 mmoles3-O-methyl-glucose/min/liter islet cell space in the presence of IgGfrom sera of patients with IDDM (p<0.05). These rates translate into a40% inhibition of glucose transport in the presence of IgG from patientswith IDDM.

If this inhibition is the result of an antibody effect on GLTJT-2activity, it should also be manifest on the GLUT-2-transfectedAtT20_(ins) cell line but not on the GLUT-1-transfected cells. Theexpression of GLUT-2 in this cell line confers them with glucosetransport characteristics remarkably similar to those found in isletcells. Purified IgG from the same patients with new-onset IDDM reducedthe initial rate of glucose transport in GLUT-2-expressing AtT20_(ins)cells from 15.5 mmoles 3-O-methyl-glucose/min/liter cell space to 6.2mmoles 3-O-methyl-glucose/min/liter cell space, p<0.05, (FIG. 7A, FIG.7B and FIG. 7C). This represents a 60% reduction in glucose transport inthe presence of IgG from patients with IDDM compared to uptake in thepresence of IgG from nondiabetic subjects.

Rat islet cells exhibit two kinetically distinct facilitated diffusionglucose transporter functions, a high K_(m) function ascribed to GLUT-2and a low K_(m) transport function attributed to unidentifiedtransporter. Results from a detailed kinetic analysis of the inhibitionof glucose transport into islet cells induced by diabetic IgG indicatedthat the inhibition was directed against the high K_(m) or GLUT-2mediated function. As a test of the specificity of inhibition of GLUT-2,additional measurements in GLUT-1 expressing AtT20_(ins) cells weremade. Although nontransfected AtT20_(ins) cells express GLUT-1constitutively, the GLUT-1-transfected cell line overexpresses thisprotein and exhibits a greater than 10-fold increase in the velocity ofglucose uptake which increases the accuracy of the transportmeasurement. Glucose uptake in GLUT-1-transfected AtT20_(ins) cellstreated with IgG from new-onset IDDM patients was indistinguishable fromtransport in the presence of IgG from nondiabetic individuals (FIG. 7A,FIG. 7B and FIG. 7C). These data indicate that IgG from new-onset IDDMpatients does not inhibit glucose transport in AtT20_(ins) cells thatexpress a facilitative glucose transporter other than GLUT-2.

2. Specificity of Interactions of Sera from Patients with New-Onset IDDHand Nondiabetic Patients for GLUT-2-Expressing AtT20_(ins) Cells

It was important to establish the specificity of IgG binding to intactGLUT-2-expressing AtT20_(ins) cells by performing analyses of IgGbinding to the parent AtT20_(ins) cells. Subtraction of the percentageof cells found in R₂ using the nontransfected AtT20_(ins) cell line fromthe percentage of cells found in R₂ using the GLUT-2-expressingAtT20_(ins) cell line would be expected to reflect the specific bindingof IgG to GLUT-2. Such analyses were performed for each individual serumand a positive interaction was defined as an increase in IgG bindinggreater than two standard deviations from the mean observed in thenondiabetic patient population. It was found that 29 of 31 (94%) of thenondiabetic population were negative for IgG binding to GLUT-2 while 23of 30 (77%) of sera from IDDM patients were positive (FIG. 8). Thus, 81%of negative results were from nondiabetic patients and 92% of positiveresults were from patients with IDDM (Table 2). The Youden index ofthese results gave J=0.73 (Table 2), and the level of significance ofthe separation between the two populations was p<0.0001.

                  TABLE 2                                                         ______________________________________                                        Regional Analyis of IgG Binding from Sera of                                  Nondiabetic Children and Patients with IDDM to GLUT-2-                        Expressing AtT20.sub.ins Cells after Subtractions of IgG Binding              to Montransfected AtT20.sub.ins Cells                                                   Individual with Peak Fluorescence in R.sub.2                                    >2 Standard  <2 Standard Deviation*                               Patient Group                                                                             Deviation* Shift                                                                           Shift                                                ______________________________________                                        IDDM        23/30 (77%).sup.†                                                                    7/30 (23%).sup.                                     Nondiabetic  2/31 (6%)   29/31 (94%)                                          Sensitivity 23/30 (77%)                                                       Specificity 29/31 (94%)                                                       False Positive                                                                             2/25 (8%)                                                        Rate                                                                          False Negative                                                                             7/36 (19%)                                                       Rate                                                                          Youden Index: J = 1 - (0.08 + 0.19) = 0.73                                    ______________________________________                                         *Defined as an increase in the peak number of fluorescent cells in R.sub.     fluorescence of greater than two standard deviations from the mean of the     number of fluorescent cells in R.sub.2 after treatment with sera from         nondiabetic children.                                                         .sup.† p < 0.0001 compared to the raondiabetic population         

EXAMPLE IV PERFUSION OF A COLUMN CONTAINING CGT-6 CELLS FOR INCREASEDINSULIN PRODUCTION

A. Methods

Insulin secretion from CGT-6 (GLUT-2 expressing AtT-20_(ins)) cells wasevaluated using a column perfusion technique (Knudsen et al., 1983).Cells were grown in liquid culture on microcarrier beads (InvitroGen).Approximately 50×10⁶ cells were harvested by gentle centrifugation (500rpm in a Sorvall RT6000B desk top centrifuge), resuspended in 4 mlKrebs-Ringer salt (KRS) solution, pH 7.4, and loaded onto a PharmaciaP10/10 column. A cell count was obtained immediately before loading thecolumn in the following manner. An aliquot of cells was taken, the beadsdigested with 1.2 U/ml Dispase (Boehringer Mannheim), the cell clumpswere dispersed by extrusion through a 25 gauge needle and the cells werecounted directly.

After the beads settled in the column, the top plunger of the column wasgently inserted and the whole apparatus was submerged in a 37° C. waterbath. The cells were then perifused as described below.

B. Results

In early secretion studies, a static incubation procedure was used inwhich cells were grown in tissue culture dishes and exposed tosecretragogue-containing media over relatively long time periods (3hours). While this technique was found to be valuable for screening newcell lines, it provides no information concerning the dynamics ofinsulin release. Perifusion experiments were therefore carried out toaddress this concern, and to evaluate whether glucose-stimulated insulinsecretion from GLUT-2 expressing AtT-20_(ins) cells occurs in a similartime frame as the rapid islet β-cell response.

Native AtT-20_(ins) cells, as well as GLUT-2 and GLUT-1 transfectedlines were grown in liquid culture on microcarrier beads (InvitroGen),harvested into a Pharmacia P10/10 column, and washed with HBSS lackingglucose for 15 minutes. The capacity of lines CGT-6 (GLUT-2transfected), CGT1-15 (GLUT-1 transfected) and the parental AtT-20_(ins)cells to secrete insulin in response to glucose was compared (FIG. 9).

Perifusion with HBSS lacking glucose was continued after the 15 minutewash-out for an additional 25 minutes (FIG. 9, Phase 1). During thisperiod, there was a gradual decline in insulin release from all threecell lines. Phase 1I was initiated by switching to HBSS buffercontaining 5 mM glucose. A 10-fold increase in insulin release fromCGT-6 cells was noted in the first sample, collected in the first 2.5minutes after the switch to glucose-containing buffer (FIG. 9). Thisincrease was sustained in 2 samples (representing a total of 5 minutes),after which insulin secretion declined to a second plateau that was3-fold above the pre-glucose level. This biphasic pattern of insulinrelease is similar to that observed upon glucose stimulation of normalislets. Only small changes in insulin release were observed in phase IIfor either the parental AtT-20_(ins) cells or the GLUT-1 transfectedCGT1-15 line.

After 25 minutes of perifusion with 5 mM glucose, the cells wereswitched back to HBSS lacking glucose (FIG. 9, Phase III). Insulinsecretion from the CGT-6 cells persisted at the glucose-stimulated levelfor approximately 10 minutes after the switch to buffer lacking glucose,but then declined rapidly. The low level of insulin release fromparental AtT-20ins cells and CGT1-16 cells was further reduced duringperifusion with glucose free media. In phase IV, cells were switchedback to buffer containing 5 mM glucose. The CGT-6 cells again showed amuch stronger secretory response to glucose, but the response was lessrapid (requiring 15 minutes to reach maximum), and was without anobvious first phase and second phase.

Switching back to buffer lacking glucose in phase V again resulted in adramatic albeit delayed reduction in insulin release from CGT-6 cells.In the last 25 minute phase (FIG. 9, phase VI), cells were perifusedwith HBSS containing the combination of 5 mhM glucose and 0.5 μMforskolin. In keeping with the results from earlier static incubationexperiments, GLUT-2 expressing CGT-6 cells exhibited a stronger insulinsecretory response to glucose+forskolin than either the parental cellsor the GLUT-1 transfected cells. The response of line CGT-6 toglucose+forskolin was sustained until the end of the experiment,suggesting that the cells were not depleted of insulin during theperifusion experiment. Consistent with this interpretation, no changesin insulin content were noticed in any of the cell lines isolated beforeand after perifusion experiments.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the composition, methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspAlaTrpGluGluGluThrGluGlySerAlaHisIleVal                                    1510                                                                          __________________________________________________________________________

I claim:
 1. A method for producing insulin comprising:(a) culturing acell capable of secreting insulin, the cell comprising a hexokinase IVgene, an insulin gene and a GLUT-2 gene, wherein at least one of saidgenes is a recombinant gene introduced into the cell by means of arecombinant vector; (b) stimulating said cell to secrete insulin; and(c) collecting the secreted insulin.
 2. The method of claim 1, whereinsaid cell is stimulated to secrete insulin by contacting said cell witha non-glucose secretagogue.
 3. The method of claim 1, wherein said cellis grown in contact with a solid support.
 4. The method of claim 3,wherein said solid support is gelatin beads.
 5. The method of claim 3,wherein said cell is contained within a column.
 6. The method of claim1, wherein the insulin produced is human insulin.
 7. The method of claim1, wherein at least a portion of the insulin produced is encoded by arecombinant insulin gene.
 8. The method of claim 2, wherein said cell iscontacted with said non-glucose secretagogue and with glucose.
 9. Themethod of claim 8, wherein said cell is simultaneously contacted withsaid non-glucose secretagogue and glucose.
 10. The method of claim 8,wherein said cell is contacted with glucose prior to contact with saidnon-glucose secretagogue.
 11. The method of claim 2, wherein saidnon-glucose secretagogue is forskolin, dibutyryl cAMP orisobutylmethylxanthine (IBMX).
 12. The method of claim 11, wherein saidnon-glucose secretagogue is forskolin.
 13. The method of claim 11,wherein said non-glucose secretagogue is dibutyryl cAMP.
 14. The methodof claim 11, wherein said non-glucose secretagogue is IBMX.
 15. Themethod of claim 1, wherein said GLUT-2 gene is a recombinant gene. 16.The method of claim 15, wherein said GLUT-2 gene is a rat islet GLUT-2gene.
 17. The method of claim 15, wherein said GLUT-2 gene comprises acDNA.
 18. The method of claim 1, wherein said cell is further defined asa cell derived from a cell capable of forming secretory granules. 19.The method of claim 18, wherein said cell is further defined as a cellderived from an endocrine cell.
 20. The method of claim 19, wherein saidcell is further defined as a cell derived from a pituitary cell or athyroid cell.
 21. The method of claim 18, wherein said cell is furtherdefined as a cell derived from an AtT-20 cell.
 22. The method of claim21, wherein said cell is further defined as a CTG-5 cell or a CTG-6cell.
 23. The method of claim 22, wherein said cell is further definedas a CTG-6 cell.
 24. The method of claim 18, wherein said cell isfurther defined as a cell derived from a GH-1 cell or a GH-3 cell. 25.The method of claim 18, wherein said cell is further defined as a cellderived from a β cell.
 26. The method of claim 18, wherein said cell isfurther defined as a cell derived from a βTC, RIN or HIT cell.
 27. Themethod of claim 1, wherein said cell expresses recombinant glucokinase.28. The method of claim 27, wherein said cell comprises a recombinantglucokinase cDNA gene.
 29. The method of claim 1, wherein said cell isfurther defined as having a reduced hexokinase activity relative to thecell line from which it was prepared.
 30. A method for producinginsulin, the method comprising:(a) culturing a cell capable of secretinginsulin in response to a non-glucose secretagogue, the cell comprising ahexokinase IV gene, an insulin gene and a GLUT-2 gene, wherein at leastone of said genes is a recombinant gene introduced into the cell bymeans of a recombinant vector; (b) contacting said cell with a buffercontaining a non-glucose secretagogue for a period of time sufficient tostimulate insulin secretion; and (c) collecting the secreted insulin.31. The method of claim 30, wherein the insulin produced is humaninsulin.
 32. A method for producing human insulin, the methodcomprising:(a) obtaining a cell that secretes human insulin in responseto a non-glucose secretagogue, the cell comprising a hexokinase IV gene,a human insulin gene and a GLUT-2 gene, wherein at least one of saidgenes is a recombinant gene introduced into the cell by means of arecombinant vector; (b) growing said cell in culture; (c) contactingsaid cell with a buffer containing a non-glucose secretagogue for aperiod of time sufficient to stimulate said cell to secrete humaninsulin; (d) collecting the media that has been in contact with saidcell; and (e) purifying human insulin from the collected media.
 33. Amethod for producing human insulin, the method comprising:(a) obtaininga population of cells that secrete human insulin in response to anon-glucose secretagogue, said cells containing at least about 1 mUnitof human insulin per million cells and comprising a human insulin geneand a GLUT-2 gene, wherein at least one of said genes is a recombinantgene introduced into the cell by means of a recombinant vector; (b)growing said cells in culture; (c) contacting said cells with a buffercontaining a non-glucose secretagogue for a period sufficient to allowsaid cells to respond; (d) collecting the media that has been in contactwith said cells; and (e) purifying human insulin from the collectedmedia.
 34. A method for producing human insulin, the methodcomprising:(a) obtaining a population of cells that secrete humaninsulin in response to a non-glucose secretagogue, the cells comprisinga human insulin gene and a GLUT-2 gene, wherein at least one of saidgenes is a recombinant gene introduced into the cell by means of arecombinant vector; (b) growing said cells in culture; (c) contactingsaid cells with a buffer containing a non-glucose secretagogue for aperiod sufficient to allow said cells to respond; (d) collecting themedia that has been in contact with said cells, said media containing atleast about 5 Units of human insulin per liter of media; and (e)purifying human insulin from the collected media.
 35. The method ofclaim 1, wherein said cell is stimulated to secrete insulin bycontacting said cell with glucose.
 36. The method of claim 33, whereinsaid cells further comprise a hexokinase IV gene.
 37. The method ofclaim 34, wherein said cells further comprise a hexokinase IV gene.